Nucleic acid sequence measurement method

Abstract

The present invention provides a nucleic acid sequence measurement method for measuring a target RNA having a specific nucleic acid sequence included in a cell suspension by hybridization, the nucleic acid sequence measurement method including: a step for heating a cell suspension at 100-200°C in a pressurized state and obtaining an RNA extract; a step for adding a proteolytic enzyme to the RNA extract, carrying out a reaction therebetween, and preparing a sample solution; a step for supplying the sample solution to a device for nucleic acid sequence measurement, the device being equipped with a fluorescent probe that hybridizes with the target RNA; a step for hybridizing the target RNA and the fluorescent probe; and a step for measuring the fluorescence from the device for nucleic acid sequence measurement.

Description

[Technical Field]

[0001] The present invention relates to a method for measuring a nucleic acid sequence. [Background technology]

[0002] A widely known method for measuring a target having a specific nucleic acid sequence contained in a sample is to use a DNA microarray (a detection probe having a complementary sequence to a specific nucleic acid sequence is provided on a solid phase surface such as a substrate). This method measures the target by utilizing the property that the target contained in a sample added to the DNA microarray is captured by the detection probe of the DNA microarray through a hybridization reaction. This method can measure not only whether or not the target is contained in the sample, but also the amount of the target contained in the sample. Patent Document 1 discloses a measurement method for measuring a target using a DNA microarray.

[0003] In order to subject a target to DNA microarray, it is necessary to extract the target nucleic acid. When the target is nucleic acid contained in a microorganism such as a bacterium, known methods for extracting nucleic acid include melting the cell membrane of the microorganism by enzymatic or chemical treatment, or disrupting the microorganism by physical stimulation using a French press or a bead beater. Patent Document 2 also discloses a method for extracting nucleic acid by heating at high temperature in a sealed container without using enzymatic or chemical treatment or physical treatment. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2015-43702 [Patent Document 2] Japanese Patent Application Laid-Open No. 2012-157265 Summary of the Invention [Problem to be solved by the invention]

[0005] If a sample containing extracted target (RNA) could be measured directly using the nucleic acid sequence measurement method of Patent Document 1 without any processing, it would be possible to significantly reduce the effort and time required for target measurement. However, if a sample containing target is directly applied to the nucleic acid sequence measurement method of Patent Document 1, there is a problem that contaminants such as proteins contained in the sample affect the hybridization reaction, degrading the measurement accuracy. In addition, the sample solution may become cloudy, making it impossible to detect the fluorescence emitted from the DNA microarray. Furthermore, during fluorescence measurement, the contaminant proteins (hereinafter also referred to as contaminant proteins) are excited by excitation light and emit fluorescence, which increases the background light.

[0006] The present invention has been made in consideration of the above circumstances, and aims to provide a nucleic acid sequence measurement method that can measure RNA having a specific sequence contained in a sample solution with high accuracy while reducing the effort and time required for measurement. [Means for solving the problem]

[0007] In order to achieve the above object, the present invention employs the following configuration. [1] A nucleic acid sequence measurement method for measuring target RNA having a specific nucleic acid sequence contained in a cell suspension by hybridization, comprising the steps of: heating the cell suspension under pressure at 100 to 200°C to obtain an RNA extract; adding a protease to the RNA extract to cause a reaction and prepare a sample solution; supplying the sample solution to a nucleic acid sequence measurement device equipped with a fluorescent probe that hybridizes with the target RNA; causing a hybridization reaction between the target RNA and the fluorescent probe; and measuring the fluorescence from the nucleic acid sequence measurement device. [2] The nucleic acid sequence measuring method described in [1], characterized in that in the step of measuring the fluorescence, the fluorescence from the nucleic acid sequence measuring device is measured without washing the sample solution supplied to the nucleic acid sequence measuring device. [3] The nucleic acid sequence measurement method according to [1] or [2], wherein in the step of obtaining the RNA extract, the cell suspension is heated in a sealed container. [4] The method for measuring a nucleic acid sequence according to any one of [1] to [3], wherein the protease is proteinase K. [5] The nucleic acid sequence measuring device comprises: a fluorescent probe having a binding portion and a base end, and a fluorescent molecule attached to a tip or intermediate position; a quenching probe having a binding portion and a base end, and a quenching molecule attached to a position adjacent to the fluorescent molecule of the fluorescent probe when the quenching probe binds to the fluorescent probe; and a substrate having a solid-phase surface to which the base end of the fluorescent probe and the base end of the quenching probe are respectively fixed, wherein the binding portion of the fluorescent probe and the binding portion of the quenching probe have complementary sequences, and at least one of the fluorescent probe and the quenching probe has a detection portion having a sequence complementary to the nucleic acid sequence of the target RNA, and the detection portion detects the target RNA. the base ends of the fluorescent probe and the quencher probe are fixed to a solid-phase surface in such a positional relationship that, when hybridization between the target RNA and the detection unit does not occur, the bond between the binding portion of the fluorescent probe and the binding portion of the quencher probe is maintained, thereby quenching the fluorescence of the fluorescent molecule by the quencher molecule that has approached the fluorescent molecule; and, when hybridization between the target RNA and the detection unit occurs, the bond between the binding portion of the fluorescent probe and the binding portion of the quencher probe is dissolved, thereby causing the fluorescent molecule that is away from the quencher molecule to exhibit fluorescence. [Effects of the Invention]

[0008] The nucleic acid sequence measurement method of the present invention has the effect of reducing the time and effort required for measurement while enabling highly accurate measurement of RNA having a specific sequence contained in a sample solution. [Brief explanation of the drawings]

[0009] [Figure 1] 1 is a flowchart showing an example of a nucleic acid sequence measurement method of the present invention. [Figure 2] 1 is a diagram showing an example of the configuration of a nucleic acid sequence measurement device used in a nucleic acid sequence measurement method of the present invention. [Figure 3] FIG. 2 is a diagram illustrating an example of the configuration of a probe. [Figure 4] FIG. 1 is a diagram schematically illustrating the principle of detecting a target RNA. [Figure 5] FIG. 1 is a diagram illustrating an example of the configuration of a nucleic acid sequence detection device. [Figure 6] FIG. 1 shows the results of electrophoresis of the fragment lengths of RNA fragments obtained after heating at 140° C. to extract RNA in Experimental Example 1. [Figure 7] FIG. 1 shows the results of electrophoresis of the fragment lengths of RNA fragments obtained after heating at 160° C. to extract RNA in Experimental Example 1. [Figure 8] 10 is a graph showing the measurement of the spot light intensity exhibited by a substrate onto which fluorescent molecules have been dropped when a protease is added and a reaction is carried out in Experimental Example 2. [Figure 9] 10 is a graph showing the measurement of the spot light intensity exhibited by a substrate onto which fluorescent molecules were dropped in Experimental Example 2 when no protease was added. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, a nucleic acid sequence measurement method according to an embodiment of the present invention will be described in detail with reference to the drawings. First, an outline of the embodiment of the present invention will be described, and then the details of the embodiment of the present invention will be described.

[0011] 〔overview〕 An embodiment of the present invention enables highly accurate measurement of RNA having a specific sequence contained in a sample solution while reducing the effort and time required for measurement. The method disclosed in the above-mentioned Patent Document 1 measures a target using a nucleic acid sequence measurement device (DNA microarray) provided with, as detection probes, a fluorescent probe to which a fluorescent molecule is attached and a quenching probe to which a quenching molecule that quenches the fluorescence of the fluorescent molecule is attached. This method makes it possible to measure the target without attaching fluorescent molecules to the target or washing the DNA microarray (to remove uncaptured targets, etc.).

[0012] In this method, a nucleic acid sequence measurement device uses a DNA microarray to measure the presence or amount of a specific nucleic acid. When a target is not present, the independent fluorescent probe and quencher probe remain bound via the binding site, and the fluorescence of the fluorescent molecule is quenched by the quencher molecule. When a target is supplied, the target binds to the detection site, the binding between the fluorescent probe and quencher probe via the binding site is dissolved, and the quencher molecule separates from the fluorescent molecule, causing the fluorescent molecule to emit fluorescence. This nucleic acid sequence measurement device can be used to measure the target contained in a sample.

[0013] The nucleic acid extraction method disclosed in the above-mentioned Patent Document 2 is a nucleic acid extraction method comprising the steps of introducing a cell suspension into a container, sealing the container, and heating the cell suspension contained in the container to a specified maximum temperature of 100°C or higher while the container is sealed.

[0014] However, when a sample containing a target is directly applied to the nucleic acid sequence measurement method of Patent Document 1, there is a problem that contaminants such as proteins contained in the sample affect the hybridization reaction, degrading the measurement accuracy. Furthermore, the sample solution may become cloudy, making it impossible to detect the fluorescence emitted from the DNA microarray. Furthermore, during fluorescence measurement, the contaminant proteins are excited by excitation light and emit fluorescence, which increases the background light.

[0015] [Embodiment] The nucleic acid sequence measurement method of this embodiment is a nucleic acid sequence measurement method for measuring target RNA having a specific nucleic acid sequence contained in a cell suspension by hybridization. The method includes the steps of: heating the cell suspension under pressure at 100 to 200°C to obtain an RNA extract; adding a protease to the RNA extract and reacting it to prepare a sample solution; supplying the sample solution to a nucleic acid sequence measurement device equipped with a fluorescent probe that hybridizes with the target RNA; hybridizing the target RNA with the fluorescent probe; and measuring fluorescence from the nucleic acid sequence measurement device. This reduces the effort and time required for measurement, suppresses the influence of contaminating proteins, and enables highly accurate measurement of the target RNA contained in the sample solution. Furthermore, it is possible to prevent the sample solution from becoming cloudy during the hybridization reaction, which would otherwise cause a decrease in fluorescence intensity. Furthermore, it is possible to reduce background light caused by the contaminating proteins.

[0016] The nucleic acid sequence measurement method of this embodiment will be described below. Fig. 1 is a flow chart showing an example of the nucleic acid sequence measurement method of the present invention. The nucleic acid sequence measurement method of the present invention will be described below with reference to Fig. 1. First, a cell suspension is heated under pressure at 100 to 200° C. to obtain an RNA extract (Step S1). There are no particular limitations on the cells from which RNA is extracted, and examples include microbial cells.

[0017] Examples of microorganisms include reeds. Ne Tobacter (Acin eTobacter spp., Actinomyces spp., Aerococcus spp., Aeromonas spp., Alclaigenes spp., Bacillus spp., Bacteriodes spp., Bordetella spp., Branhamella spp., Brevibacterium spp., Campylobacter spp., Candida spp., Capnocytophaga Capnocytophagia spp., Chromobacterium spp., Clostridium spp., Corynebacterium spp., Cryptococcus spp., Deinococcus spp., Enterococcus spp., Erysielothrix spp., Escherichia spp., Flavobacterium spp., Gemella spp. Lactobacillus species, Haemophilus species, Klebsiella species, Lactobacillus species, Lactococcus species, Legionella species, Leuconostoc species, Listeria species, Micrococcus species, Mycobacterium species, Neisseria species, Cryptosporidium species, Nocardia species, Oerskovia species, Paracoccus species, Pediococcus species, Peptostreptococcus species, Propionibacterium species, Proteus species, Pseudomonas species, Rahnella species, Rhodococcus species, Rhodospirillium species,Examples of such bacteria include Staphlococcus species, Streptomyces species, Streptococcus species, Vibrio species, and Yersinia species. The present invention can also be applied to animal cells, insect cells, plant cells, mycoplasma, viruses, and the like other than microorganisms. The cells of the present invention may be a mixture of multiple different types of cells as described above. Furthermore, there are microorganisms that take the form of spores or spores in poor nutritional conditions, and the state of cells depending on such growth conditions is not important.

[0018] A cell suspension can be obtained by suspending cells in a culture medium, a buffer solution, or the like. The cell suspension may be a culture medium in which cells have been cultured, or a culture medium in which cells have been cultured suspended in a buffer solution. The buffer solution used here is one used to stabilize and improve the efficiency of RNA extraction, and examples of buffer solutions that can be used include Tris-HCl buffer, phosphate buffer, sodium borate buffer, SSC buffer (Saline Sodium Citrate Buffer), SSPE buffer (Saline Sodium Phosphate-EDTA Buffer), and carbonate-bicarbonate buffer.

[0019] Next, the cell suspension is heated under pressure at 100 to 200°C to obtain an RNA extract. The method for heating the cell suspension at 100 to 200°C under pressure is not particularly limited, as long as it can be heated to 100 to 200°C at a pressure equal to or higher than atmospheric pressure. For example, a method in which the cell suspension is placed in a container, the container is sealed, and then the container is heated at 100 to 200°C may be used. The heating temperature may be 100 to 200°C and is determined appropriately depending on the type of cells to be used, but 120 to 165°C is preferred, 120 to 160°C is more preferred, and 140 to 160°C is particularly preferred. By setting the heating temperature at 100 to 200°C, the target RNA can be made to have a uniform and suitable fragment length. Heating temperatures below 100°C may result in a decrease in the efficiency of target RNA extraction from cells. Furthermore, the fragment length of the target RNA may be too long to bind to a fluorescent probe. Furthermore, if the heating temperature exceeds 200°C, the fragment length of the target RNA may become too short, which may reduce the efficiency of hybridization to the fluorescent probe. The pressure is not particularly limited as long as it is equal to or higher than atmospheric pressure, but is preferably 100 to 1500 kPa, and more preferably 200 to 700 kPa. A pressure of 100 to 1500 kPa allows the target RNA to be fragmented to a more uniform and suitable length. If the pressure is less than 100 kPa, the efficiency of extracting the target RNA from cells may decrease. Furthermore, the fragment length of the target RNA may be too long to bind to the fluorescent probe. Furthermore, if the pressure exceeds 1500 kPa, the fragment length of the target RNA may become too short, resulting in a decrease in the efficiency of hybridization to the fluorescent probe.

[0020] The container for containing the cell suspension is not particularly limited as long as it has a mechanical structure for sealing and is heat-resistant, and examples thereof include boil-lock type tubes, heat-resistant resin tubes, metal containers, glass containers, etc. The heating means is not particularly limited as long as it can be heated to 100 to 200°C, and examples thereof include dry heat blocks, oil baths, microwaves, direct flame heating, steam heating, etc.

[0021] The heating time is not particularly limited and can be set appropriately depending on the type of cells to be applied, the amount of cell suspension, etc., but is preferably 20 seconds to 10 minutes, more preferably 30 seconds to 5 minutes, and may be, for example, 40 seconds to 3 minutes, 1 to 2 minutes, etc.

[0022] For example, when the cells are microbial cells, an example of heating conditions suitable for gram-negative bacteria is a heating time of 15 seconds or more and less than 2 minutes, and a heating temperature of 105 to 125°C or less. For example, when the cells are microbial cells, an example of heating conditions suitable for gram-positive bacteria and fungi is a heating time of 1 minute or less and a heating temperature of 125 to 160°C or less.

[0023] When a sealed container is used to create a pressurized state, the volume of the container capable of uniformly heating the cell suspension in a short time is preferably 2 ml or less, more preferably 0.6 ml or less, and particularly preferably 0.2 ml or less.

[0024] The cell suspension may be cooled after heating. The cooling method after heating may be rapid cooling or natural cooling. Rapid cooling methods include, for example, rapidly cooling the heated cell suspension together with the container using a Peltier element. Rapid cooling of the cell suspension generates a thermal shock, improving cell disruption efficiency and thus RNA extraction efficiency. This method is effective for cells with a rigid structure, such as gram-positive bacteria and fungi. The natural cooling method includes leaving the container containing the heated cell suspension at room temperature and allowing it to cool naturally. When the cell suspension is cooled naturally, no heat shock is applied, so RNA can be extracted while minimizing damage to the RNA. This method is effective when extracting long-chain RNA.

[0025] After heating, the cell suspension is released from the pressurized state. The pressurized state may be released while the cell suspension is still pressurized, or after the pressure has been reduced to atmospheric pressure. Examples of methods for releasing the pressurized state include releasing a sealed container that is kept at a high temperature of 100 to 200°C. When a sealed container that is kept at a high temperature of 100 to 200°C is released, the internal pressure at the time of release is equal to or greater than atmospheric pressure, resulting in a sudden change in pressure. This generates shear forces, which improves cell disruption efficiency and RNA extraction efficiency. This method is effective for cells with rigid structures, such as gram-positive bacteria and fungi.

[0026] Examples of methods for releasing the pressurized state after reducing it to atmospheric pressure include a method in which a container in a high temperature state of 100 to 200°C is cooled to 100°C or below and then the sealed state of the container is released. When a container in a high temperature state of 100 to 200°C is cooled to 100°C or below and then the sealed state of the container is released, no shear force is applied when the container is released, and therefore RNA can be extracted with reduced damage to the RNA. This method is effective when extracting long-chain RNA.

[0027] A cell lysis promoter may be used when extracting RNA from a cell suspension. The use of a cell lysis promoter enables efficient RNA extraction from cells with a rigid structure, such as Gram-positive bacteria and fungi, and from cells in a more robust state, such as spores and oocysts. The cell lysis promoter may be added either before or after high-temperature treatment.

[0028] When a cell lysis promoter is added to the sample before high-temperature treatment, the cell membrane structure is weakened by the promoter, allowing the high-temperature treatment to work effectively and improving RNA extraction efficiency. High-temperature treatment in the cell lysis promoter can effectively exert its effect. This is effective when the sample amount is small and more reliable extraction under a single condition is desired.

[0029] When a cell lysis promoter is added to a sample after high-temperature treatment, the cell membrane structure is weakened by the high-temperature treatment, allowing the cell lysis promoter to act effectively, allowing nucleic acids to be extracted highly efficiently in a short period of time. This is effective for cells with a strong structure.

[0030] Examples of cell lysis promoters include alkalis (e.g., NaOH, KOH), acids (e.g., HCl, H2SO4), enzymes (proteases such as proteinase K, polysaccharide-degrading enzymes such as chitinase, lysozyme, and zymolyase), surfactants (anionic surfactants such as SDS, cationic surfactants such as CTAB (cetyltrimethylammonium bromide), nonionic surfactants such as Triton-X, and zwitterionic surfactants such as betaine (a general term for compounds with a specific structure, e.g., trimethylglycine)), redox agents (e.g., hydrogen peroxide, β-mercaptoethanol, and dithiothreitol), protein denaturants (e.g., guanidine hydrochloride and urea), and chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid)). A mixture of these agents may also be used. A buffer solution may also be added, if necessary.

[0031] When a sealed container is used to create a pressurized state, controlling the conditions inside the container can expedite the transition to a high-temperature, pressurized state. Specifically, one method involves introducing a cell suspension into the container so that no bubbles or air spaces remain, sealing the container, and then proceeding to the heating step. Furthermore, for cell suspensions with a small volume relative to the container's internal volume, layering a high-boiling-point solvent such as mineral oil over the air space can improve the sealability. In this case, the container is also heated after sealing. In this case, the vapor fills the gas phase, and heating to a temperature above the boiling point is performed after the vapor pressure is reached. This allows the container to be pressurized more quickly, allowing the temperature to reach 100-200°C.

[0032] When a sealed container is used to create a pressurized state, mechanical sealing of the container can prevent the sealed state from being released when the internal pressure of the container increases, thereby enabling stable heat treatment. An example of mechanical sealing is a method of pressing down on the container with a member of a shape that fits the container (insertion).

[0033] Next, a protease is added to the RNA extract and reacted to prepare a sample solution (step S2). The protease is not particularly limited as long as it can decompose the cell-derived contaminant proteins contained in the RNA extract, and examples thereof include proteinase K, actinase E, trypsin, chymotrypsin, and pepsin. These proteases may be used alone or in combination.

[0034] This step decomposes cell-derived contaminating proteins contained in the RNA extract using protease. This suppresses the effects of the contaminating proteins. Furthermore, this step also suppresses the clouding of the sample solution, which would otherwise occur due to a reaction between the contaminating proteins and the salt used in the hybridization reaction in the nucleic acid sequence measurement device described below. Furthermore, in the step of measuring fluorescence from the nucleic acid sequence measurement device described below, the contaminating proteins are prevented from being excited by excitation light and exhibiting fluorescence, thereby reducing background light.

[0035] The amount of protease to be added to the RNA extract is not particularly limited as long as it is an amount that can decompose contaminating cell-derived proteins, and is, for example, a concentration that results in a final concentration in the reaction solution of 20 to 500 μg / mL.

[0036] The reaction temperature between the contaminating proteins in the RNA extract and the protease is appropriately determined based on the optimal temperature of the protease used, for example, 30 to 40° C. in the case of proteinase K. The reaction time is appropriately determined based on the properties of the protease used, for example, 10 to 20 minutes in the case of proteinase K.

[0037] Next, the sample solution is supplied to a nucleic acid sequence measurement device equipped with a fluorescent probe that hybridizes with the target RNA (step S3). A salt is added to the sample solution to hybridize the target RNA having a specific nucleic acid sequence with the fluorescent probe. Examples of the salt to be added include sodium chloride. Instead of the salt, a buffer containing a salt may be added. Examples of the buffer containing a salt include SSC buffer, Church's phosphate buffer, and SSPE buffer.

[0038] The concentration of the salt to be added is preferably, for example, a concentration that results in a 2- to 10-fold dilution of the final concentration, and more preferably a concentration that results in a 5-fold dilution.

[0039] An example of a nucleic acid sequence measurement device that supplies a sample solution is the nucleic acid sequence measurement device disclosed in Japanese Patent Application Laid-Open No. 2015-43702. Specifically, the nucleic acid sequence measuring device comprises: a fluorescent probe having a binding portion and a base end, and a fluorescent molecule attached to a tip or intermediate position; a quencher probe having a binding portion and a base end, and a quencher molecule attached to a position adjacent to the fluorescent molecule of the fluorescent probe when the quencher probe binds to the fluorescent probe; and a substrate having a solid-phase surface to which the base ends of the fluorescent probe and the quencher probe are fixed, wherein the binding portion of the fluorescent probe and the binding portion of the quencher probe have complementary nucleic acid sequences, and at least one of the fluorescent probe and the quencher probe has a nucleic acid sequence complementary to the nucleic acid sequence of the target RNA. The base ends of the fluorescent probe and the quencher probe are fixed to a solid-phase surface in such a positional relationship that, when hybridization between the target RNA and the detection unit does not occur, the binding between the binding portion of the fluorescent probe and the binding portion of the quencher probe is maintained, thereby quenching the fluorescence of the fluorescent molecule by the quencher molecule approaching the fluorescent molecule; when hybridization between the target RNA and the detection unit occurs, the binding between the binding portion of the fluorescent probe and the binding portion of the quencher probe is dissolved, thereby causing the fluorescent molecule, separated from the quencher molecule, to exhibit fluorescence. Therefore, when hybridization between the target RNA and the detection unit does not occur, the binding between the binding portion of the fluorescent probe and the binding portion of the quencher probe is maintained. As a result, the fluorescence of the fluorescent molecule is quenched by the quencher molecule approaching the fluorescent molecule. When hybridization between the target RNA and the detection unit occurs, the binding between the binding portion of the fluorescent probe and the binding portion of the quencher probe is dissolved. This causes the fluorescent molecules separated from the quenching molecules to emit fluorescence. This nucleic acid sequence measurement device fixes the base ends of the fluorescent probe and quencher probe, which are independent molecules, allowing the quenching effect to be properly exerted, improving detection sensitivity. It also eliminates the need for a labeling process and the need for a washing process.

[0040] FIG. 2 is a diagram showing an example of the configuration of a nucleic acid sequence measurement device used in the nucleic acid sequence measurement method of the present invention, and FIG. 3 is a diagram showing an example of the configuration of a probe.

[0041] As shown in Figures 2 and 3, the nucleic acid sequence measurement device is composed of a fluorescent probe 10 having a fluorescent molecule 11 attached to the complementary sequence of a target RNA 30 to be detected, and a quenching probe 20 having a quenching molecule 21 attached, immobilized on a solid-phase surface 100 such as a substrate.

[0042] In the present invention, the principle of quenching by fluorescence resonance energy transfer is used. The fluorescent molecule used in the present invention is not particularly limited as long as it is a molecule that emits fluorescence when excited by specific excitation light, and examples thereof include EDANS, Coumarin, FAM, FITC, Cy2, TF2, TF3, HEX, JOE, TET, Cy3, Cy5, Alexa Fluor (registered trademark) 532, Alexa Fluor (registered trademark) 610, Alexa Fluor (registered trademark) 647, ATTO532, ATTO633, Qdot (registered trademark) 565, Qdot (registered trademark) 585, Qdot (registered trademark) 605, Qdot (registered trademark) 705, and iFluor TM 532, iFluor TM Known substances such as 647 can be used.

[0043] The quenching molecule used in the present invention is not particularly limited, and examples thereof include Dabcyl, TQ1, TQ2, TQ3, Eclipse (registered trademark), BHQ1, BHQ2, BHQ3, Cy5Q, Cy7Q, Iowa Black (registered trademark) FQ, Iowa Black (registered trademark) RQ, IRDye QC-1, QSY7, QSY21, and QXL57. 0 etc. The following known substances can be used.

[0044] There are no particular limitations on the combination of fluorescent molecules and quencher molecules, and examples thereof include a combination of EDANS, Coumarin, or TF2 with Dabcyl or TQ1, a combination of FAM, FITC, TET, Alexa Fluor (registered trademark) 532, Cy2, Cy3, TF2, or TF3 with TQ2, a combination of Alexa Fluor (registered trademark) 532, Cy3, HEX, JOE, TF2, TF3, TF4, or TET with TQ3, a combination of Alexa Fluor (registered trademark) 532, TF2, Cy3, FAM or HEX with Eclipse (registered trademark), a combination of Alexa Fluor (registered trademark) 532, TF2, TF3, Cy3, FAM, HEX, TET, or Cy3 with BHQ1, a combination of TF3, TF4, Cy3, Cy5, or HEX with BHQ2, Cy5, Alexa Fluor (registered trademark) 647, TF5 with Iowa Black (registered trademark) RQ, IRDye, etc. combinations of Cy3, TF3, TF4 and Cy5Q, Iowa Black (registered trademark) FQ, Iowa Black (registered trademark) RQ, IRDye QC-1, QSY7 or QXL570; combinations of Alexa Fluor (registered trademark) 532 and Cy5Q, TQ2, TQ3, Iowa Black (registered trademark) FQ, Iowa Black (registered trademark) RQ, IRDye QC-1, QSY7 or QXL570; combinations of TF3 and BHQ1, BHQ2 or Cy5Q.

[0045] The substrate used in the present invention may be a plate-shaped material such as quartz, glass, silicon, single crystals such as calcium fluoride and sapphire, ceramics, or a resin material, which has a rectangular shape when viewed from above. Examples of resin materials include COP (cycloolefin polymer), COC (cyclic olefin copolymer), polycarbonate, acrylic resin, and polyethylene resin, which have excellent optical properties and chemical and thermal stability. The substrate may have any shape when viewed from above.

[0046] As shown in FIG. 3 , the fluorescent probe 10 includes an X portion 12 of several bases that is provided at the 3′ end and is a complementary sequence of the target RNA 30, a detection sequence 13 that is provided following the X portion 12 and is a complementary sequence of the target RNA 30, and a linker 14 that is connected to the detection sequence 13 and continues to the 5′ end. A fluorescent molecule 11 is fixed to the 3′ end of the fluorescent probe 10.

[0047] The quenching probe 20 comprises a Y portion 22 of several bases provided from the 5' end, a detection sequence 23 provided following the Y portion 22 and having a complementary sequence to the target RNA 30, and a linker 24 connected to the detection sequence 23 and extending to the 3' end, and a quenching molecule 21 is fixed to the 5' end of the quenching probe 20.

[0048] The fluorescent probe 10 and the quencher probe 20 are immobilized on a solid-phase surface 100 via linkers 14 and 24, respectively. The sequence of the X portion 12 of the fluorescent probe 10 and the sequence of the Y portion 22 of the quencher probe 20 are complementary to each other. The fluorescent probe 10 and the quencher probe 20 are immobilized at positions where the X portion 12 of the fluorescent probe 10 and the Y portion 22 of the quencher probe 20 can bind to each other, and a positional relationship is ensured such that when the X portion 12 of the fluorescent probe 10 and the Y portion 22 of the quencher probe 20 bind to each other, the quencher molecule 21 approaches the fluorescent molecule 11, thereby causing the fluorescent molecule 11 to enter a quenched state.

[0049] Furthermore, it is desirable to design the affinity between the fluorescent probe 10 and the target RNA 30 to be higher than the affinity between the fluorescent probe 10 and the quencher probe 20 due to the X portion 12 and the Y portion 22 .

[0050] In the present invention, "complementary" means that one nucleic acid sequence has a nucleic acid sequence that can form a double-stranded state with another nucleic acid sequence, and does not necessarily have to be completely complementary, and may contain some mismatched base pairs. Furthermore, the fluorescent molecule or quencher molecule does not have to be attached to the tip of the probe, and the fluorescent molecule or quencher molecule may be attached to a position midway along the probe.

[0051] The nucleic acid sequence measuring device used in the nucleic acid sequence measuring method of the present invention can be produced as follows.

[0052] (1) Solution preparation First, a probe solution is prepared by mixing the fluorescent probe 10 and the quenching probe 20, and the probe concentration is adjusted.

[0053] (2) Coupling Next, the probe solution is heated and then rapidly cooled to couple the fluorescent probe 10 with the quencher probe 20. This causes the fluorescent probe 10 to bind with the quencher probe 20 via the X portion 12 and the Y portion 22. Here, for example, the probe solution is heated to 95°C, maintained at that temperature for 5 minutes, and then rapidly cooled to 25°C to couple the fluorescent probe 10 with the quencher probe 20.

[0054] (3) Fixation to a solid surface Next, a probe solution in which the fluorescent probe 10 and the quencher probe 20 are coupled is spotted onto the solid phase surface, and the fluorescent probe 10 and the quencher probe 20 are immobilized on the solid phase surface 100 .

[0055] (4) Cleaning Next, the solid phase surface 100 is washed to remove excess probes that have not been immobilized. By the above procedure, a nucleic acid sequence measurement device is manufactured.

[0056] In this way, the fluorescent probe 10 and the quenching probe 20 are bound to the solid-phase surface 100 while being bound to each other via the X portion 12 and the Y portion 22. This allows the positional relationship between the fluorescent probe 10 and the quenching probe 20 to be properly controlled, allowing the quenching effect to be properly exerted, thereby improving detection sensitivity.

[0057] Next, the target RNA 30 and the fluorescent probe 10 are hybridized (step S4). As shown in Figure 4, when the target RNA 30 is not present, the fluorescent molecule 11 and the quencher molecule 21 are brought into close proximity to each other by binding to the X portion 12 and the Y portion 22 (Figure 3) of several bases adjacent to the fluorescent molecule 11 and the quencher molecule 21, respectively. In this state, the fluorescent molecule 11 does not emit fluorescence due to the influence of the quencher molecule 21 even when irradiated with excitation light.

[0058] When a sample solution containing target RNA 30 is supplied to the solid-phase surface 100 of the nucleic acid sequence measurement device and a hybridization reaction is carried out, the target RNA 30 binds to the fluorescent probe 10, as shown in Figure 4. This releases the bond between the X portion 12 and the Y portion 22, increasing the distance between the quencher molecule 21 and the fluorescent molecule 11, thereby releasing the quenching state and causing the fluorescent molecule 11 to emit fluorescence when irradiated with excitation light.

[0059] Next, after the hybridization reaction, the fluorescence from the nucleic acid sequence measurement device is measured (step S5). As described above, when the target RNA 30 binds to the fluorescent probe 10 in the sample solution, the bond between the X portion 12 and the Y portion 22 is released, increasing the distance between the quencher molecule 21 and the fluorescent molecule 11. This releases the quenching state of the fluorescent molecule 11, causing the fluorescent molecule 11 to emit fluorescence upon irradiation with excitation light. The presence or absence of the target RNA 30 in the sample solution can be confirmed by measuring this fluorescence using a fluorescence reader (described below). Furthermore, the target RNA 30 contained in the sample solution can be quantified by measuring the fluorescence intensity from the nucleic acid sequence measurement device. Furthermore, since uncaptured target RNA 30 contained in the solution does not emit fluorescence, the nucleic acid sequence measurement device does not need to be washed. Therefore, it is possible to observe the solid-phase surface through the solution in the presence of the sample solution. The present invention can suppress clouding of the sample solution due to the reaction between contaminating proteins and the salt used in the hybridization reaction, and reduce background light caused by the contaminating proteins. This allows the amount of light to be measured with high accuracy without being affected by washing, and also allows real-time measurement during hybridization.

[0060] Next, the nucleic acid sequence measuring device will be described. The nucleic acid sequence measuring apparatus includes the nucleic acid sequence measuring device and a fluorescence reading device that measures the amount of fluorescence emitted from the nucleic acid sequence measuring device. 5 is an example of a configuration diagram showing a nucleic acid sequence measurement device. The nucleic acid sequence measurement device acquires images of the nucleic acid sequence measurement device 60 before and after binding of the target RNA 30 and the fluorescent probe 10. After acquiring the image before binding, the temperature of the solid-phase surface of the nucleic acid sequence measurement device 60 is raised by the temperature control stage 46 to allow the binding reaction to proceed, and then the temperature is lowered again to room temperature, after which an image after binding is acquired.

[0061] In order to promote the binding between the target RNA 30 and the fluorescent probe 10, it is preferable that the temperature-controlled stage 46 has a stirring function using shaking, rotation of a nucleic acid sequence measurement device, a vortex mixer, or the like during the reaction between the target RNA 30 and the fluorescent probe 10.

[0062] In the optical system of the fluorescence reader 40, laser light emitted from a laser light source 41 passes through a mirror 45 and is reflected by a dichroic mirror 44, irradiating the solid phase of the nucleic acid sequence measurement device. The irradiated light becomes excitation light 33 for fluorescent molecules 11 on the solid phase surface of the nucleic acid sequence measurement device 60, and the fluorescent molecules 11 enter an excited state, causing them to emit fluorescence 34.

[0063] Fluorescence emitted from the solid phase surface of the nucleic acid sequence measurement device 60 passes through a dichroic mirror 44 and passes through an imaging optical system 43, where a fluorescent image is formed and detected on the detection element of a CCD camera 42. In order to prevent the excitation light 33 from leaking into the fluorescence 34, a bandpass filter matched to the excitation light wavelength may be installed on the excitation light 33 side, or a bandpass filter matched to the fluorescence wavelength to be detected may be installed on the fluorescence 34 side.

[0064] The fluorescence images obtained by the nucleic acid sequence measurement device can capture images before and after binding of the target RNA 30 and fluorescent probe 10 in the same spot. Therefore, they are not affected by variations in light intensity between solid phases or spots. In addition, the amount of change in fluorescence can be calculated from the fluorescence images before and after the binding reaction to calculate the number of molecules that have undergone the binding reaction. The amount of change in fluorescence can be calculated using the average light intensity of the entire spot, or the amount of change in fluorescence for each pixel of the spot image.

[0065] The nucleic acid sequence measuring device may be equipped with a computer for controlling the CCD camera 42, an arithmetic unit for calculating the light intensity of the image, and a recording unit for storing the image, the light intensity, and the like.

[0066] The nucleic acid sequence measurement device detects fluorescence from the side opposite the immobilization surface of the detection spot on the solid phase surface, so a fluorescence microscope, confocal microscope, evanescent fluorescence detection device, thin film oblique illumination microscope, sheet illumination microscope, structured illumination microscope, multiphoton excitation microscope, etc. can be used.

[0067] The scope of application of the present invention is not limited to the above-described embodiments, and the present invention can be widely applied to nucleic acid sequence measurement methods that measure target RNA having a specific nucleic acid sequence contained in a sample by hybridization. [Example]

[0068] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0069] (Experimental Example 1) RNA extraction from Staphylococcus aureus Staphylococcus aureus (NBRC100910) was cultured in SCD medium for 1 day, and 1 mL of the resulting culture was centrifuged at 5000 × g to remove the supernatant. The resulting cells were suspended in 2.50 μL of lysis solution (50 mM Tris-HCl buffer (pH 8.0), 0.1% SDS), placed in a sealed container, and heated at 140°C or 160°C for 45 seconds to obtain an RNA extract. To the resulting RNA extract, 2 μL of DNase (trade name: Recombinant DNase I (RNase-free); manufactured by Takara Bio Inc.; concentration: 5 U / μL) was added and the mixture was incubated at 37°C for 10 to 30 minutes to decompose and remove DNA. The length of the RNA fragments was then confirmed by electrophoresis. The results of heating at 140°C and 160°C are shown in Figure 6 and Figure 7, respectively.

[0070] As shown in Figure 6, it was confirmed that heating at 140°C resulted in uniform fragmentation into strands of a length suitable for hybridization. Furthermore, as shown in Figure 7, it was confirmed that heating at 160°C resulted in fragmentation into strands of a shorter length, thereby further improving the efficiency of hybridization.

[0071] (Experimental Example 2) Detection of RNA extracted from E. coli using a nucleic acid sequence measurement device E. coli (NBRC3972) was cultured in SCD medium for 1 day, and 1 mL of the culture was centrifuged at 5,000 × g to remove the supernatant. The resulting cells were suspended in 2.50 μL of lysis solution (composition: 50 mM Tris-HCl buffer (pH 8.0), 0.1% SDS), placed in a sealed container, and heated at 140°C for 45 seconds to obtain an RNA extract. To the obtained RNA extract, 5 μL (final concentration: 500 μg / mL) of proteinase K (manufactured by Takara Bio Inc.) was added, and the mixture was incubated at 37° C. for 15 minutes. Next, SSC buffer was added to a final concentration of 5 times diluted, and the solution was applied to a microarray on which a fluorescent probe capable of detecting E. coli was immobilized. The microarray was incubated at 60°C for 3 hours, and the intensity of the light spot from the microarray was measured using a fluorescence reader. The results are shown in Figure 8. As a control, the RNA extract obtained above was similarly supplied to the microarray without adding proteinase K, and the spot light intensity was measured using a fluorescence reader. The results are shown in Figure 9. In Figures 8 and 9, NTC indicates the results when a sample solution not containing the target E. coli was supplied to the microarray.

[0072] As shown in Figure 8, when proteinase K was added to the RNA extract, the background light was low in the absence of target RNA, and the increase in spot light intensity was large in the presence of target RNA. In contrast, as shown in Figure 9, when proteinase K was not added to the RNA extract, the background light was high in the absence of target RNA, and the increase in spot light intensity was small in the presence of target RNA. From the above results, it was confirmed that adding a proteolytic enzyme to the RNA extract reduces the background light of the microarray and increases the amount of light emitted when target RNA is present, enabling accurate detection of RNA from cell extracts. [Explanation of symbols]

[0073] 10 Fluorescent Probes 11 Fluorescent molecules 12 X part (joint part) 13 Detection array (detection section) 14 Linker (proximal end) 20 Quenching Probes 21 Quenching molecules 22 Y part (joint part) 23 Detection array (detection section) 24 Linker (proximal end) 30 Target RNA 60 Nucleic acid sequence measurement device 40 Fluorescence reader 41 Laser light source 42 CCD cameras 43 Imaging optical system 44 Dichroic Mirror 45 Mirror 46 Temperature Control Stage 100 Solid surface

Claims

1. A nucleic acid sequence measurement method for measuring target RNA having a specific nucleic acid sequence contained in a cell suspension by hybridization, comprising: heating the cell suspension under pressure at 100 to 200°C to obtain an RNA extract; a step of adding a protease that decomposes cell-derived contaminant proteins contained in the RNA extract to the RNA extract from which the RNA has been extracted, and reacting the protease to prepare a sample solution; supplying the sample solution to a nucleic acid sequence measurement device equipped with a fluorescent probe that hybridizes with the target RNA; a step of hybridizing the target RNA with the fluorescent probe; measuring fluorescence from the nucleic acid sequence measurement device; A method for measuring a nucleic acid sequence comprising:

2. 2. The nucleic acid sequence measuring method according to claim 1, wherein the step of measuring the fluorescence measures the fluorescence from the nucleic acid sequence measuring device without washing the sample solution supplied to the nucleic acid sequence measuring device.

3. 3. The nucleic acid sequence measurement method according to claim 1, wherein in the step of obtaining the RNA extract, the cell suspension is heated in a sealed container.

4. 4. The method for measuring a nucleic acid sequence according to claim 1, wherein the protease is proteinase K.

5. The nucleic acid sequence measuring device is a fluorescent probe having a binding portion and a base end and having a fluorescent molecule attached to the tip or intermediate position; a quencher probe having a binding portion and a base end, and having a quencher molecule attached to a position adjacent to the fluorescent molecule of the fluorescent probe when the quencher probe is bound to the fluorescent probe; a substrate having a solid phase surface on which the proximal end of the fluorescent probe and the proximal end of the quenching probe are respectively fixed; Equipped with the binding portion of the fluorescent probe and the binding portion of the quenching probe have complementary sequences; at least one of the fluorescent probe and the quenching probe has a detection part having a sequence complementary to a nucleic acid sequence of the target RNA; When hybridization between the target RNA and the detection unit does not occur, the binding between the binding unit of the fluorescent probe and the binding unit of the quenching probe is maintained, and the fluorescence emitted by the fluorescent molecule is quenched by the quenching molecule that has come close to the fluorescent molecule, the proximal ends of the fluorescent probe and the quencher probe are immobilized on a solid-phase surface in such a positional relationship that, when hybridization between the target RNA and the detection unit occurs, the binding between the binding unit of the fluorescent probe and the binding unit of the quencher probe is dissolved, causing the fluorescent molecule separated from the quencher molecule to exhibit fluorescence; The method for measuring a nucleic acid sequence according to any one of claims 1 to 4.

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