JP7133867B2 - NUCLEIC ACID DETECTION METHOD AND NUCLEIC ACID DETECTION DEVICE - Google Patents

Description

The present invention relates to a nucleic acid detection method and a nucleic acid detection device.

In genetic testing and the like, methods for specifically detecting target nucleic acids are very important in order to examine the presence of viruses, bacteria, and the like. Known methods for detecting nucleic acids include gel electrophoresis, solid-phase probes, nucleic acid chromatography, real-time PCR, and the like. For example, in Patent Document 1, a complex of a nucleic acid and a dielectric fine particle is collected on a fine electrode by dielectrophoresis, and the collected complex is electrically or optically detected. A detection method is described.

JP 2015-109826 A

The nucleic acid detection method disclosed in Patent Document 1 requires the use of electrical equipment or optical equipment, which is costly in terms of cost and time. Therefore, there has been a demand for a simple method that can further reduce costs.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a nucleic acid detection method and a nucleic acid detection device that can easily detect nucleic acids.

The present inventors have found that when a complex is formed between a nucleic acid and a magnetic particle in a solvent, and the solvent is replaced with a solvent having a low salt concentration, the magnetic particle with the nucleic acid bound and the magnetic particle with the non-nucleic acid bound. , found that the dispersion state differs when magnetic force is applied to the magnetic particles, and completed the present invention.

The method for detecting nucleic acid according to the first aspect of the present invention comprises
forming a complex between the nucleic acid and the magnetic particles in an aqueous sodium chloride solution ;
a step of detecting the nucleic acid based on a change in the state of dispersion of the complex when a magnetic force is applied to the complex in salt-free water ;
Including .

In this case, the complex is a complex of a biotin-labeled nucleic acid and a magnetic particle having a biotin-binding protein.
You can do it.

Further, the step of detecting the nucleic acid includes a step of visually determining the presence or concentration of the nucleic acid,
You can do it.

Further, in the step of forming the complex, the nucleic acid is amplified and a complex is formed between the amplified nucleic acid and the magnetic particles,
You can do it.

The nucleic acid detection device according to the second aspect of the present invention comprises
a container containing a complex of nucleic acid and magnetic particles and an aqueous sodium chloride solution ;
a magnet that exerts a magnetic force on the composite;
a supply for supplying salt-free water to the container;
a detection unit that detects the nucleic acid based on a change in the dispersed state of the complex when a magnetic force is applied to the complex in the container to which the water is supplied;
Prepare .

According to the present invention, nucleic acids can be easily detected.

FIG. 2 is a diagram showing a nucleic acid detection device according to Embodiment 2 of the present invention; FIG. 10 is a diagram showing a nucleic acid detection device according to Embodiment 3 of the present invention; 3 is a block diagram showing the hardware configuration of a detection unit in the nucleic acid detection device shown in FIG. 2. FIG. 3 is a diagram showing a flowchart of nucleic acid detection processing by the nucleic acid detection device shown in FIG. 2. FIG. FIG. 2 is a diagram showing the state of dispersion of magnetic particles in a hydrophobic container before bringing a magnet close to it in Example 1; (A) and (B) show the dispersed state of nucleic acid-unbound magnetic particles and nucleic acid-magnetic particle complexes, respectively. FIG. 10 is a diagram showing the state of dispersion of magnetic particles in the hydrophobic container after 2.5 seconds have passed since the magnet was brought closer in Example 1; (A) and (B) show the dispersed state of nucleic acid-unbound magnetic particles and nucleic acid-magnetic particle complexes, respectively. FIG. 5 is a diagram showing the state of dispersion of magnetic particles in the hydrophobic container after five seconds have passed since the magnet was brought closer in Example 1; (A) and (B) show the dispersed state of nucleic acid-unbound magnetic particles and nucleic acid-magnetic particle complexes, respectively. FIG. 10 is a diagram showing the state of dispersion of magnetic particles in the hydrophobic container after 13.5 seconds have passed since the magnet was brought closer in Example 1; (A) and (B) show the dispersed state of nucleic acid-unbound magnetic particles and nucleic acid-magnetic particle complexes, respectively. FIG. 10 is a diagram showing the dispersed state of magnetic particles on a hydrophilic substrate before a magnet is brought closer in Example 2; (A) and (B) show the dispersed state of nucleic acid-unbound magnetic particles and nucleic acid-magnetic particle complexes, respectively. FIG. 10 is a diagram showing the state of dispersion of magnetic particles on the hydrophilic substrate after 15 seconds have passed since the magnet was brought closer in Example 2; (A) and (B) show the dispersed state of nucleic acid-unbound magnetic particles and nucleic acid-magnetic particle complexes, respectively. FIG. 10 is a diagram showing the state of dispersion of magnetic particles in a hydrophobic container before bringing a magnet close to it in Comparative Example 1; (A) and (B) show the dispersed state of nucleic acid-unbound magnetic particles and nucleic acid-magnetic particle complexes, respectively. FIG. 10 is a diagram showing the state of dispersion of magnetic particles in the hydrophobic container after 15 seconds have passed since the magnet was brought closer in Comparative Example 1; (A) and (B) show the dispersed state of nucleic acid-unbound magnetic particles and nucleic acid-magnetic particle complexes, respectively. FIG. 10 is a diagram showing a state of dispersion of magnetic particles on a hydrophilic substrate before a magnet is brought closer in Comparative Example 2; (A) and (B) show the dispersed state of nucleic acid-unbound magnetic particles and nucleic acid-magnetic particle complexes, respectively. FIG. 10 is a diagram showing the state of dispersion of magnetic particles on the hydrophilic substrate after 15 seconds have passed since the magnet was brought closer in Comparative Example 2; (A) and (B) show the dispersed state of nucleic acid-unbound magnetic particles and nucleic acid-magnetic particle complexes, respectively. FIG. 10 is a diagram showing the dispersed state of magnetic particles on the hydrophilic substrate after several seconds have passed since the magnet was brought closer in Example 3;

DETAILED DESCRIPTION OF THE INVENTION Embodiments for carrying out the present invention will be described in detail below. However, the present invention is not limited to the following embodiments.

(Embodiment 1)
A nucleic acid detection method according to Embodiment 1 includes a formation step and a detection step. In the forming step, complexes of nucleic acids and magnetic particles are formed in solvent A (first solvent).

As used herein, the term "nucleic acid" refers to a chain polymer compound in which nucleotide units containing pentose, phosphoric acid and base are linked. The nucleic acid may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). A nucleic acid may be a single-stranded polymeric compound or a double-stranded polymeric compound. Nucleic acids can be, for example, cDNA, mRNA, RNA, cRNA, and the like.

The nucleic acid may be nucleic acid in the sample to be detected or may be pre-amplified nucleic acid. In the forming step, the nucleic acid may be amplified and a complex between the amplified nucleic acid and the magnetic particles may be formed. That is, in the forming step, amplification of the nucleic acid and formation of the complex between the nucleic acid and the magnetic particles may be performed simultaneously. The method for amplifying nucleic acid is not particularly limited as long as it can amplify the nucleic acid. -Based Amplification (NASBA) method, Loop-Mediated Isothermal Amplification (LAMP) method, and the like.

Solvent A is not particularly limited as long as it has a higher salt concentration than solvent B, which will be described later.

The magnetic particles need only have the property of responding to a magnetic field, and may contain, for example, metals such as iron, manganese, nickel, cobalt, chromium and platinum. The size of the magnetic particles is not particularly limited, and may be, for example, 0.1 to 10 μm. The size of the magnetic particles is preferably 1 to 5 μm from the viewpoint of binding to nucleic acids.

The complex of nucleic acid and magnetic particles may be formed by integrating the nucleic acid and the magnetic particles. A complex can be formed, for example, by directly or indirectly binding a nucleic acid to be detected and a magnetic particle. The method for forming the complex may be, for example, any one of the following methods (i) to (vii).

(i) a method of forming a complex by specifically binding a chemical substance that modifies the end of a nucleic acid to a magnetic particle; (ii) chemically binding a nucleic acid probe to the surface of a magnetic particle; A method of forming a complex by hybridizing with a nucleic acid (iii) without modifying the end of the nucleic acid with a chemical substance, by binding to magnetic particles via a phosphate group or a hydroxyl group, etc. A method of forming a body (iv) A method of forming a complex by binding a chemical substance to a part of a nucleic acid (for example, a non-terminal part) and binding the chemical substance to magnetic particles (v) Physical adsorption Alternatively, a method of forming a complex using chemisorption (vi) A chemical substance that recognizes a specific sequence or structure of a nucleic acid and binds to the nucleic acid is bound or adsorbed to the magnetic particles to form a complex. (vii) a method of forming a complex by synthesizing nucleic acids on magnetic particles

Examples of the method (iv) include a method using a restriction enzyme that recognizes a base sequence, and a method that uses a substance that recognizes a double-stranded structure such as a fluorescent dye (intercalator, minor/major groove binder, etc.). The method of utilization, etc. are mentioned. That is, the above method (iv) can form a complex by using a combination of a nucleic acid and a restriction enzyme with magnetic particles and an anti-restriction enzyme antibody.

In method (v) above, for example, magnetic particles to which nucleic acids physically or chemically adsorb can be used as the magnetic particles. In the above method (v), for example, a complex between the nucleic acid and the magnetic particles can be formed by binding the nucleic acid to a chemical substance that physically or chemically adsorbs to the magnetic particles.

The above method (vii) is not limited to a mode in which the amplified nucleic acid is bound to the magnetic particles after the nucleic acid is amplified, and may be a mode in which the nucleic acid is amplified and the nucleic acid is synthesized on the magnetic particles at the same time. . For example, one of the primers used for PCR is immobilized on magnetic particles, and nucleic acid is synthesized so that the primer is extended during PCR. The method for amplifying nucleic acids is not limited to the PCR method, and other methods for amplifying nucleic acids such as the LAMP method may be used.

In the forming step, when nucleic acid amplification and complex formation are performed simultaneously, for example, magnetic particles can be added to a PCR solution to synthesize nucleic acids on the magnetic particles. One of the primers used in the PCR method is immobilized on the magnetic particles so that the nucleic acid is synthesized on the magnetic particles by the nucleic acid synthesis reaction. Thereby, a complex of nucleic acid and magnetic particles can be produced at the same time as the nucleic acid is amplified. In the formation step, amplification of the nucleic acid and formation of the complex are carried out at the same time, so that time can be saved, and the nucleic acid can be detected in a shorter period of time.

The forming step may be performed, for example, for 15 minutes, may be performed for 10 minutes, or may be performed for 1 to 5 minutes.

The complex may be a complex of a biotin-labeled nucleic acid and a magnetic particle with a biotin-binding protein. Biotin-binding proteins include, for example, avidin, streptavidin and neutravidin. Biotin-labeled nucleic acids form complexes with magnetic particles via biotin-avidin binding.

In the detection step, the nucleic acid is detected based on changes in the state of dispersion of the complex when a magnetic force is applied to the complex in solvent C (second solvent) having a salt concentration lower than that of solvent A. For example, in the detection step, the complex obtained in the formation step is separated using a magnet and added to solvent C. Subsequently, a magnetic force may be applied to the complex in solvent C. A method of applying a magnetic force to the composite includes, for example, a method of bringing a magnet closer to the composite. Examples of such a method include a method of bringing a magnet closer to a container containing a solvent containing a complex, a method of bringing a magnet closer to a substrate onto which a solvent containing a complex has been dropped, and the like.

Solvent C is not particularly limited as long as it has a lower salt concentration than solvent A. For example, it may be pure water, ion-exchanged water, distilled water, or TE buffer (Tris/EDTA solution). or the like to reduce the salt concentration.

Solvent C may be obtained by replacing at least part of Solvent A with Solvent B having a lower salt concentration than Solvent A while exerting a magnetic force on the complex in Solvent A. Solvent B is not particularly limited as long as it has a lower salt concentration than solvent A. For example, it may be pure water, ion-exchanged water, distilled water, or TE buffer (Tris/EDTA solution). or the like to reduce the salt concentration. Part or all of solvent A may be substituted with solvent B.

The method of obtaining the solvent C is not limited to the method of replacing at least part of the solvent A with the solvent B, and may be a method of adding the solvent B to the solvent A, or the like. Solvent C having a lower salt concentration than Solvent A can be obtained even by such a method. In this case, the magnitude relationship of the salt concentration is "solvent A>solvent C>solvent B". Since "solvent A>solvent C", the nucleic acid can be detected based on the change in the state of dispersion of the complex in solvent C.

When replacing at least part of the solvent A with the solvent B in the preparation of the solvent C, by applying a magnetic force to the complex in the solvent A, the complex aggregates due to the action of the magnetic force. Solvent B can be substituted for A.

Changes in the state of dispersion of the composite can be determined visually. That is, the detection step may include a step of visually determining the presence or concentration of the nucleic acid. For example, in the step of determining the nucleic acid concentration, when the complex aggregates in the vicinity of the magnet, the concentration is determined by comparing the color density of the aggregated portion of a sample with an unknown nucleic acid concentration with reference data. be able to. The reference data prepared in advance includes the color density or photograph of the aggregated portion for each sample obtained by serially diluting the nucleic acid concentration.

Changes in the state of dispersion of the composite in the solvent include, for example, a phenomenon in which the composite dispersed in the solvent agglomerates on the magnet in the solvent; and a phenomenon in which the complex is adsorbed to the surface of the substrate when the solvent in which the complex is dispersed is dripped onto the surface of the substrate.

According to the nucleic acid detection method according to the present embodiment, when a magnetic force is applied to the complex of the nucleic acid and the magnetic particles in the solvent C having a lower salt concentration than the solvent A in which the complex was formed, Nucleic acids can be detected based on changes in the state of dispersion of complexes. As a result, nucleic acids can be visually detected without using electrical equipment or optical equipment. Therefore, nucleic acids can be detected simply and quickly.

(Embodiment 2)
Next, the nucleic acid detection device 100 according to Embodiment 2 will be described. FIG. 1 schematically shows the configuration of a nucleic acid detection device 100 . The detection device 100 includes a container 10 containing a complex 11 of nucleic acid and magnetic particles and a solvent 12 (first solvent), a container 20 containing a solvent 22, and a magnet 30 applying a magnetic force to the complex 11. , and a pipe 40 for supplying the solvent 22 contained in the container 20 to the container 10 .

Container 10 may be a hydrophobic container or a hydrophilic container. A hydrophobic container may be, for example, a container comprising a material of polypropylene, polytetrafluoroethylene, polyethylene, polydimethylsiloxane, or silicone rubber. The hydrophilic container may be, for example, a container containing a material such as glass. The container 10 may be made of a material used for a hydrophilic container whose surface has undergone a hydrophobic treatment, or a material used for a hydrophobic container whose surface has been given a hydrophilic treatment. For example, hydrophobization includes a method of treating the glass surface with a silane coupling agent (methyltrimethoxysilane, tetraethoxysilane, etc.), and hydrophilization includes a method of treating the glass surface with oxygen plasma or the like. be.

The solvent 12 is, for example, a solvent having a salt concentration higher than that of the solvent 22 described below. The solvent 12 may be, for example, an aqueous sodium chloride solution, an aqueous lithium chloride solution, or various Good's buffer solutions.

Container 20 contains solvent 22 (eg, deionized water). The detection device 100 is configured to be able to supply the solvent 22 from the container 20 to the container 10 . That is, the solvent 22 is supplied from the container 20 to the container 10 via the pipe 40 . The method of supplying the solvent 22 is not particularly limited, and the pipe 40 may be used as the supply unit as in the present embodiment. Alternatively, the solvent 22 in the container 20 may be poured directly into the container 10 . In this case, the second container 20 becomes the supply unit.

At least a portion of solvent 12 may be removed from container 10 prior to supplying solvent 22 to container 10 . As a result, a sufficient amount of the solvent 22 is supplied to the container 10, the salt concentration in the solvent in the container 10 is sufficiently lowered, and the nucleic acid can be detected with high accuracy. At least part of the solvent 12 may be drawn out from the container 10 while applying a magnetic force to the composite 11 with the magnet 30 . This can facilitate the derivation operation of the solvent 12 .

Container 20 may be a hydrophobic container or a hydrophilic container. Hydrophobic containers may be, for example, containers comprising polypropylene, polytetrafluoroethylene, polyethylene, polydimethylsiloxane, or silicone rubber materials. The hydrophilic container may be, for example, a container containing a material such as glass. The container 20 may be made of a material used for a hydrophilic container whose surface has been hydrophobized, or a material used for a hydrophobic container whose surface has been given a hydrophilic treatment. For example, hydrophobization includes a method of treating the glass surface with a silane coupling agent (methyltrimethoxysilane, tetraethoxysilane, etc.), and hydrophilization includes a method of treating the glass surface with oxygen plasma or the like. be.

Deionized water refers to water obtained by a deionization process. Deionized water can be, for example, water from which ions have been removed using an ion exchange resin. Deionized water may be, for example, ultrapure water (resistivity: 15 MΩ·cm or more), pure water (resistivity: 0.1 to 15 MΩ·cm), tap water, or the like.

Magnet 30 is used to apply a magnetic force to composite 11 . The magnet 30 is not particularly limited as long as it can apply magnetic force. The shape of the magnet 30 may be disc-shaped or rod-shaped. Magnet 30 is positioned adjacent container 10 on the exterior of container 10 . Thereby, the magnetic force can be effectively applied to the composite 11 .

According to the detection device 100, in the container 10, the solvent 22 is supplied through the pipe 40, so that a magnetic force can be applied to the complex 11 in the solvent whose salt concentration is lower than that of the solvent 12. . Thereby, the user can detect the nucleic acid based on the change in the dispersed state of the complex 11 . Therefore, the detection device 100 can easily detect nucleic acids without using electrical equipment, optical equipment, or the like.

Magnet 30 may be immersed in solvent 12 in container 10 . Further, for example, the container 10 may be provided with a discharge portion for discharging the solvent 12 . The description of the nucleic acid detection device 100 can be appropriately applied to the nucleic acid detection method. In addition, the description of the nucleic acid detection method can also be appropriately applied to the nucleic acid detection device 100 .

When nucleic acid amplification and complex 11 formation are performed simultaneously in container 10, the supply unit may supply reagents such as primers, enzymes, and deoxynucleoside triphosphates necessary for nucleic acid amplification to container 10. good.

(Embodiment 3)
The nucleic acid detection device 200 according to the third embodiment will be mainly described in terms of differences from the detection device 100 according to the second embodiment. As shown in FIG. 2 , the detection device 200 further includes a detection unit 50 in addition to the configuration of the detection device 100 .

As shown in FIG. 3, the detection unit 50 includes a camera 1, a storage unit 2, a RAM (Random Access Memory) 3, an input device 4, a display device 5, and a CPU (Central Processing Unit) 6. Prepare. The camera 1, storage unit 2, RAM 3, input device 4, display device 5 and CPU 6 are interconnected via a bus 7 so as to be communicable.

The camera 1 captures an image of the dispersed state of the composite 11 inside the container 10 . The storage unit 2 includes non-volatile storage media such as ROM (Read Only Memory), HDD (Hard Disk Drive), and flash memory. The storage unit 2 stores a detection program 8 in addition to various data and software programs.

RAM3 functions as a main memory of CPU6. When the detection program 8 is executed by the CPU 6 , the detection program 8 is developed in the RAM 3 . Image data input from the camera 1 is temporarily stored in the RAM 3 .

The input device 4 is hardware for the user to input data to the detector 50 . When the user inputs through the input device 4 to execute the process of detecting nucleic acid, the input device 4 inputs data instructing the CPU 6 to execute the process. The display device 5 is a display for displaying the results of detection by the detection unit 50 and the like.

The CPU 6 reads the detection program 8 stored in the storage unit 2 into the RAM 3 and executes the detection program 8, thereby realizing the functions of the detection unit 50 described below.

The detection unit 50 detects nucleic acids based on changes in the dispersion state of the complexes 11 when a magnetic force is applied to the complexes 11 in a solvent having a salt concentration lower than that of the solvent 12 in the container 10 . For example, according to data input via the input device 4 that instructs execution of a process, the detection unit 50 applies a magnetic force to the complex 11 in a solvent having a salt concentration lower than that of the solvent 12. The dispersed state of the complex 11 is imaged via the camera 1 . When image data is input from the camera 1 to the RAM 3, the detector 50 analyzes the stored image data and detects nucleic acids.

In the image data analysis, the dispersion state may be determined using a known image recognition technique. For example, the detection unit 50 determines a change in the dispersed state of the complex 11 in the area corresponding to the vicinity of the magnet 30 in the image based on the luminance value of the image data. When the composite 11 aggregates near the magnet 30, the aggregate of the composite 11 appears as a dark portion in the region corresponding to the vicinity of the magnet 30 in the image. The detection unit 50 determines that the complexes 11 are aggregated when the brightness value of the region is less than the threshold, and detects that the container 10 contains nucleic acids. When the complex 11 does not aggregate near the magnet 30 and the magnetic particles not bound to the nucleic acid aggregate near the magnet 30, the detection unit 50 determines that there is no aggregation if the luminance value of the region is equal to or greater than the threshold value. , and it is detected that the container 10 contains a nucleic acid. The threshold value of the luminance value is stored in the storage unit 2, and the detection unit 50 refers to the threshold value stored in the storage unit 2 and detects nucleic acids by analyzing the image data.

The above-described threshold value can be appropriately set by analyzing and comparing an image of the dispersed state of the magnetic particles that are not bound to the complex 11 and the nucleic acid in advance. The analysis of image data can employ a known method as well as a method using luminance values. The detection unit 50 may determine the presence or absence of aggregation of the complex 11 based on a value obtained by quantifying the brightness or darkness of the region corresponding to the vicinity of the magnet 30 in the image. In addition, instead of analyzing one image, the detection unit 50 compares the image of the container 10 captured at time t1 with the image of the container 10 captured at time t2, a predetermined time after time t1. may be used to determine the presence or absence of agglomeration.

The detection unit 50 notifies the user via the display device 5 of at least one of detection of nucleic acid and non-detection of nucleic acid.

Nucleic acid detection processing by the detection device 200 will be described with reference to FIG. The detection unit 50 waits for input of data instructing execution of processing (step S1; No). When data instructing execution of processing is input (step S1; Yes), the detection unit 50 captures an image of the dispersion state of the composites 11 when a magnetic force is applied to the composites 11 (step S2). . Subsequently, the detection unit 50 analyzes the image data (step S3). If the detection unit 50 detects that nucleic acid is contained in the container 10 (step S4; Yes), the detection unit 50 displays that the nucleic acid is detected on the display device 5 (step S5), and starts the nucleic acid detection process. finish. On the other hand, when the detection unit 50 does not detect nucleic acid in the container 10 (step S4; No), the detection unit 50 displays on the display device 5 that no nucleic acid has been detected (step S6), and detects nucleic acid. End the process.

The nucleic acid detection device 200 according to the present embodiment includes the detection unit 50, so that the nucleic acid can be easily detected without visual observation. Nucleic acids can be rapidly and easily detected in a large number of samples by analyzing changes in the state of dispersion of a plurality of samples to be detected using a multi-well plate.

Note that the detection unit 50 determines the concentration of the nucleic acid based on the change in the dispersion state of the complexes 11 when a magnetic force is applied to the complexes 11 in a solvent having a salt concentration lower than that of the solvent 12. good too. For example, when the complex 11 aggregates in the vicinity of the magnet 30, the detection unit 50 acquires the luminance value of the region corresponding to the vicinity of the magnet 30 in the image of the sample to be detected, and serially dilutes the concentration of the nucleic acid. The concentration of nucleic acid in the sample to be detected can be detected by referring to the calibration curve prepared by measuring the brightness value of each sample. Further, the detection unit 50 may detect the density based on the area of the dark portion of the region corresponding to the vicinity of the magnet 30 in the image. The calibration curve data may be stored in the storage unit 2 in advance.

Further, the detection unit 50 may display the image captured by the camera 1 on the display device 5 . By viewing the image through the display device 5, the user can confirm the change in the dispersed state, which is highly convenient.

Further, in the detection device 200, the detection unit 50 may control the supply unit and the discharge unit. For example, the supply unit comprises a dispensing system connected to the pipe 40, and the detection unit 50 supplies the container 10 with the solvent 22 sucked from the container 20 via the supply unit. In this case, when the supply unit supplies the solvent 22 from the container 20 to the container 10 , the detection unit 50 may take an image of the dispersion state of the composite 11 via the camera 1 . Further, the discharge section may include suction means, and the detection section 50 may discharge the solvent 12 from the container 10 via the discharge section. In this case, when the discharge unit discharges the solvent 12 from the container 10 , the supply unit may supply the solvent 22 from the container 20 to the container 10 .

The detection program 8 may be provided as a program that causes a computer to function as the detection unit 50 . The detection program 8 detects changes in the dispersed state of the complexes 11 when a magnetic force is applied to the complexes 11 of nucleic acid and magnetic particles formed in the solvent 12 in a solvent having a salt concentration lower than that of the solvent 12. The computer functions as a detection unit 50 that detects nucleic acids based on.

The detection program 8 and various software programs used in the detection device 200 are stored in CD-ROM (Compact Disc Read Only Memory), DVD (Digital Versatile Disc), magneto-optical disc (Magneto-Optical Disc), USB (Universal Serial Bus) memory. , memory cards, HDDs, and other computer-readable recording media for distribution. By installing the detection program 8 and various software programs in a specific or general-purpose computer, the computer can function as the detection device 200 . Alternatively, the detection program 8 and various software programs may be stored in a storage device of another server on the Internet, and the detection program 8 and various software programs may be downloaded from the server.

EXAMPLES The present invention will now be described more specifically based on examples. However, the present invention is not limited to the following examples.

[Example 1: Change in dispersion state of magnetic particles in hydrophobic container]
(Amplification of nucleic acid)
A 391 bp fungal double-stranded DNA (hereinafter also referred to as 391 bp DNA) was used as the nucleic acid. The 391 bp DNA was amplified by PCR using pUC19 DNA (manufactured by Takara Bio) as a template, excised from gel electrophoresis and purified. The primers used in the PCR method were as follows.
Primer 1: 5'-TTGCCGGGAAGCTAGAGTAA (SEQ ID NO: 1)
Primer 2: 5'-GCTATGTGGCGCGGTATTAT (SEQ ID NO: 2)

Primer 1 is modified with biotin at the 5' end. Therefore, 391 bp DNA can be bound to Streptavidin-modified magnetic particles (diameter 2.8 μm) described later via biotin-avidin binding.

(Formation of complex)
Streptavidin-modified magnetic particles (Dynabeads M-280 Streptavidin, manufactured by Life Technologies, diameter 2.8 μm, 3.25×10 particles/μl) and 391 bp DNA amplified by PCR (1.5 ng/μl=3.5×10 cells/μl) was suspended in a reaction solution (5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl). Then, the binding reaction was carried out at room temperature for 15 minutes to obtain a complex of nucleic acid and magnetic particles (hereinafter also referred to as "nucleic acid-magnetic particle complex"). Next, the nucleic acid-magnetic particle complexes were separated using a magnet, suspended in ion-exchanged water, and the ion-exchanged water containing the nucleic acid-magnetic particle complexes was placed in a polypropylene microtube.

Separately, the above-described Streptavidin-modified magnetic particles to which nucleic acid is not bound (hereinafter also referred to as “nucleic acid-unbound magnetic particles”) were suspended in ion-exchanged water, placed in a polypropylene microtube, and used as a control. board.

(Change in dispersed state due to action of magnetic force)
A magnet was brought close to the outer wall surface of each polypropylene microtube, and changes in the dispersion state of the nucleic acid-magnetic particle complex and nucleic acid-unbound magnetic particles in the deionized water due to the action of magnetic force were observed. The results are shown in Figures 5-8.

5(A) and 5(B) are photographs of nucleic acid-unbound magnetic particles in ion-exchanged water and nucleic acid-magnetic particle complexes in ion-exchanged water, respectively, before a magnet is brought close to the back of a polypropylene microtube. indicates

6(A) and 6(B) show, respectively, nucleic acid-unbound magnetic particles in ion-exchanged water and nucleic acid- A photograph of a magnetic particle composite is shown.

7(A) and 7(B) show, respectively, nucleic acid-unbound magnetic particles in ion-exchanged water and nucleic acid-magnetic particles in ion-exchanged water 5 seconds after a magnet is brought close to the back of a polypropylene microtube. A photograph of the composite is shown.

8(A) and 8(B) show, respectively, nucleic acid-unbound magnetic particles in ion-exchanged water and nucleic acid- A photograph of a magnetic particle composite is shown.

As shown in FIGS. 5 to 8, nucleic acid-unbound magnetic particles aggregated near the magnet. In contrast, the nucleic acid-magnetic particle complex did not aggregate near the magnet, but was adsorbed to the walls of the polypropylene microtube. As described above, the nucleic acid-unbound magnetic particles and the nucleic acid-magnetic particle complex differed in dispersion state when a magnetic force was applied. Therefore, it was confirmed that nucleic acids can be detected based on changes in the state of dispersion.

[Example 2: Change in dispersion state of magnetic particles on hydrophilic substrate]
Ion-exchanged water containing the nucleic acid-magnetic particle complex obtained in the same manner as in Example 1 was dropped onto a glass blood reaction plate (blood reaction plate with two holes, manufactured by Toshin Riko Co., Ltd.). The nucleic acid-unbound magnetic particles used in Example 1 were suspended in ion-exchanged water, dropped onto a glass blood reaction plate, and used as a control.

A magnet was brought close to the lower surface of the glass blood reaction plate, and changes in the dispersed state of the nucleic acid-magnetic particle complexes and nucleic acid-unbound magnetic particles in the deionized water due to the action of magnetic force were observed. The results are shown in FIGS. 9 and 10. FIG.

Figures 9(A) and 9(B) show nucleic acid-unbound magnetic particles in ion-exchanged water and nucleic acid-magnetic particle complexes in ion-exchanged water, respectively, before the magnet is brought close to the back surface of the glass blood reaction plate. Show pictures.

10(A) and 10(B) show, respectively, nucleic acid-unbound magnetic particles in ion-exchanged water and nucleic acid-magnetism in ion-exchanged water 15 seconds after the magnet was brought close to the lower surface of the glass blood reaction plate. A photograph of a particle composite is shown.

As shown in FIGS. 9 and 10, the nucleic acid-unbound magnetic particles did not aggregate in the vicinity of the magnet, but were adsorbed to the surface of the glass blood reaction plate. In contrast, the nucleic acid-magnetic particle complexes aggregated near the magnet. As described above, the nucleic acid-magnetic particle complexes and the nucleic acid-unbound magnetic particles differed in dispersion state when a magnetic force was applied. Therefore, it was shown that nucleic acids can be detected based on changes in the state of dispersion.

[Comparative Example 1: Change in dispersion state of magnetic particles in hydrophobic container]
The nucleic acid-magnetic particle complex and nucleic acid-unbound magnetic particles in the reaction solution were observed for changes in the state of dispersion due to the action of magnetic force in the same manner as in Example 1, except that they were not suspended in ion-exchanged water. The results are shown in FIGS. 11 and 12. FIG.

Figures 11(A) and 11(B) are photographs of the nucleic acid-unbound magnetic particles in the reaction solution and the nucleic acid-magnetic particle complex in the reaction solution, respectively, before the magnet is brought close to the back of the polypropylene microtube. indicates

Figures 12(A) and 12(B) show, respectively, nucleic acid-unbound magnetic particles in the reaction solution and nucleic acid-magnetic particles in the reaction solution 15 seconds after the magnet was brought close to the back of the polypropylene microtube. A photograph of the composite is shown.

As shown in FIGS. 11 and 12, both the nucleic acid-unbound magnetic particles and the nucleic acid-magnetic particle complex aggregated near the magnet. As described above, when not suspended in ion-exchanged water, there was no difference in the dispersed state between the nucleic acid-unbound magnetic particles and the nucleic acid-magnetic particle complex when a magnetic force was applied.

[Comparative Example 2: Change in dispersion state of magnetic particles on hydrophilic substrate]
The nucleic acid-magnetic particle complex and nucleic acid-unbound magnetic particles in the reaction solution were observed for changes in the state of dispersion due to the action of magnetic force in the same manner as in Example 2, except that they were not suspended in ion-exchanged water. The results are shown in FIGS. 13 and 14. FIG.

Figures 13(A) and 13(B) show the nucleic acid-unbound magnetic particles in the reaction solution and the nucleic acid-magnetic particle complexes in the reaction solution, respectively, before the magnet is brought close to the back surface of the glass blood reaction plate. Show pictures.

14(A) and 14(B) show, respectively, nucleic acid-unbound magnetic particles in the reaction solution and nucleic acid-magnetism in the reaction solution 15 seconds after the magnet was brought close to the lower surface of the glass blood reaction plate. A photograph of a particle composite is shown.

As shown in FIGS. 13 and 14, both nucleic acid-unbound magnetic particles and nucleic acid-magnetic particle complexes aggregated near the magnet. Thus, when not suspended in ion-exchanged water, the nucleic acid-magnetic particle complexes and the nucleic acid-unbound magnetic particles did not differ in dispersion state when a magnetic force was applied.

[Example 3: Detection of novel coronavirus (SARS-CoV-2)]
(Amplification of nucleic acid)
2019-nCoV_N_Positive Control (manufactured by IDT), which is the SARS-CoV-2 gene, was used as the nucleic acid. The primers used in the PCR method were as follows. The 5' end of primer F is modified with biotin.
Primer F (NIID_2019-nCOV_N_F2): 5'-AAATTTTGGGGACCAGGAAC (SEQ ID NO: 3)
Primer R (NIID_2019-nCOV_N_R2): 5'-TGGCAGCTGTGTAGGTCAAC (SEQ ID NO: 4)

GoTaq Probe qPCR Master Mix (manufactured by Promega) was used as an enzyme for PCR. Prepare a PCR reaction solution (GoTaq Master mix (1x), primer F (0.5 μM), primer R (0.7 μM), 2019-nCoV_N_Positive Control, reaction volume 20 μl), thermal cycle (2 minutes at 95 ° C., 40 cycles of 15 seconds at 95° C. and 60 seconds at 60° C. were performed.

(Formation of complex)
After completion of the thermal cycle, the PCR reaction solution was mixed with a solution containing streptavidin-modified magnetic microparticles (Dynabeads M-280 Streptavidin (manufactured by ThermoFisher Scientific)) and allowed to react at room temperature for 15 minutes. After completion of the reaction, the magnetic fine particles were resuspended in 100 μl of deionized water, and a blood reaction plate (made of soda lime glass, two holes, size: 5 mm×26 mm×76 mm, hole size: φ22×1.6 mm, 0-1841- 01, manufactured by AS ONE). The blood reaction plate was placed on a permanent magnet (NE025 neodymium φ5×5 (N40), manufactured by Niroku Seisakusho) to observe aggregation of the magnetic microparticles.

As shown in FIG. 15, when the number of SARS-CoV-2 genes added to the PCR reaction solution is 10 ⁵ to 10 ² , there is a clear difference compared to when no gene is added (non-target control; NTC). There is a difference. Aggregation of the magnetic microparticles, when containing genes, took a few seconds. From the above, it was shown that at least 100 SARS-CoV-2 can be detected using this method.

The embodiments described above are for the purpose of explaining the present invention and are not intended to limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims rather than the embodiments. Various modifications made within the scope of the claims and within the meaning of equivalent inventions are considered to be within the scope of the present invention.

The present invention is suitable for detecting nucleic acids.

1 camera, 2 storage unit, 3 RAM, 4 input device, 5 display device, 6 CPU, 7 bus, 8 detection program, 10, 20 container, 11 complex, 12, 22 solvent, 30 magnet, 40 pipe, 50 detection Part, 100, 200 detection device

Claims (5)

forming a complex between the nucleic acid and the magnetic particles in an aqueous sodium chloride solution ;
a step of detecting the nucleic acid based on a change in the state of dispersion of the complex when a magnetic force is applied to the complex in salt-free water ;
including
A method for detecting nucleic acids. The complex is a complex of a biotin-labeled nucleic acid and the magnetic particles having a biotin-binding protein,
The method of claim 1. The step of detecting the nucleic acid comprises visually determining the presence or concentration of the nucleic acid,
3. A method according to claim 1 or 2. In the step of forming the complex, the nucleic acid is amplified and a complex is formed between the amplified nucleic acid and the magnetic particles;
The method according to any one of claims 1-3 . a container containing a complex of nucleic acid and magnetic particles and an aqueous sodium chloride solution ;
a magnet that exerts a magnetic force on the composite;
a supply for supplying salt-free water to the container;
a detection unit that detects the nucleic acid based on a change in the dispersed state of the complex when a magnetic force is applied to the complex in the container to which the water is supplied;
comprising
Nucleic acid detector.

Images