Nucleic acid detection method and detection reagent

Abstract

To provide a technique of promptly detecting RNA.SOLUTION: The present invention provides a detection kit for detecting a target nucleic acid from a fluid containing the target nucleic acid. The target nucleic acid is RNA. The kit includes a first nucleic acid cleavage enzyme and a second nucleic acid cleavage enzyme. The first nucleic acid cleavage enzyme is 5'-Nuclease, and the second nucleic acid cleavage enzyme is FEN-1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a nucleic acid detection method, and more particularly to a method for detecting the presence or absence of a nucleic acid to be detected in the presence of a detection nucleic acid capable of forming a complex with the nucleic acid sample to be detected and a nucleic acid cleaving enzyme. This application claims priority based on Japanese Patent Application No. 2017-148402, filed on July 31, 2017, the contents of which are incorporated herein by reference. [Background technology]

[0002] Many methods exist for accurately and quickly detecting and quantifying DNA in genetic diagnosis (Patent Documents 1-3, Non-Patent Documents 1 and 2). While there are methods for detecting and quantifying RNA, these require laborious and time-consuming procedures such as reverse transcription. Furthermore, reverse transcription reactions are prone to bias. Therefore, the use of isothermal amplification reactions is considered to enable RNA detection in a short time with minimal manipulation. Among these, Invasive Cleavage Assay (ICA), which uses nucleic acid-cleaving enzymes such as flapendonuclease 1 (FEN-1) and 5'-nuclease, is an effective method due to its ease of use and reaction stability.

[0003] However, ICA using FEN-1 recognizes the invasion structure on the DNA / DNA duplex and cleaves the flap site, but only a small amount of cleavage occurs in the case of an RNA / DNA duplex. On the other hand, ICA using 5'-nuclease, which is also a nuclease-cleaving enzyme, can recognize the invasion structure on the RNA / DNA duplex and cleave the flap site, but its cleavage activity is reduced in the case of a DNA / DNA duplex. In addition, the flap cleavage activity of 5'-nuclease in the case of a DNA / DNA duplex is significantly inferior to that of FEN-1. Therefore, if you want to cleave both flap sites in a reaction system that contains a mixture of DNA / DNA duplexes and RNA / DNA duplexes, using only FEN-1 or 5'-nuclease will result in a decrease in overall cleavage activity.

[0004] For example, when it is desired to efficiently amplify a detection signal in a detection reaction targeting a certain nucleic acid, a method that combines two types of cleavage reactions is used. That is, in the first step, a flap is cleaved, and in the second step, the cleaved flap and the detection nucleic acid that forms the invasive structure are cleaved, and the cleavage products are detected. After the second step reaction is completed, the flap can be reused in the first or second step reaction, so signal amplification is faster than detecting the flap in the first step. In this case, if the target of detection is DNA and the detection nucleic acid is also DNA, detection is possible using ICA using only FEN-1. However, if the target of detection is RNA, whether the detection nucleic acid is DNA or RNA, the overall cleavage activity decreases due to the characteristics of each enzyme described above, and the time required for detection increases. Furthermore, although 5'-nuclease has flap cleavage activity against the aforementioned RNA / DNA, side reactions due to misrecognition by the enzyme occur. Therefore, increasing the amount of enzyme in an attempt to shorten the detection time increases the frequency of side reactions, making it difficult to accurately interpret the detection results.

[0005] To improve this situation, a method has been reported in which the first and second steps, which are conventionally carried out in the same reaction solution, are separated (Non-Patent Documents 3 and 4). Specifically, a reaction solution is prepared in which only the first step proceeds, using 5'-nuclease as the nucleic acid cleavage enzyme, target RNA as the nucleic acid, and two types of oligo DNA necessary for forming the invasion structure. After the first step has proceeded to a certain extent, a detection nucleic acid is added as the second step, and an oligo RNA called an arrester oligo is also added at the same time, thereby suppressing side reactions due to misrecognition by the enzyme. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 4016050 [Patent Document 2] Patent No. 4362150 [Patent Document 3] Patent No. 4363988 [Non-patent literature]

[0007] [Non-Patent Document 1] The Journal of Biological Chemistry, 1999, 274, 21387-21394 [Non-patent document 2] The Journal of Biological Chemistry, 2000, 275, 24693-24700 [Non-patent document 3] RNA, 2003, 9, 1552-1561 [Non-patent document 4] RNA, 2004, 10, 1153-1161 Summary of the Invention [Problem to be solved by the invention]

[0008] However, opening the reaction vessel midway not only complicates the procedure but also increases the risk of false positives due to external sample contamination, potentially compromising the accuracy of the test. Therefore, when rapid and accurate RNA detection is required, a procedure that is simple to operate, maintains cleavage activity even in a reaction system where both structures are present, and suppresses side reactions is desirable.

[0009] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for rapidly detecting RNA. [Means for solving the problem]

[0010] The nucleic acid detection method of the present invention is a nucleic acid detection method for detecting a target nucleic acid from a fluid containing the target nucleic acid, and includes a detection reagent mixing step of mixing a detection reagent comprising a first nucleic acid cleaving enzyme and a second nucleic acid cleaving enzyme with the fluid, and the first flap site associated with a first invasion structure that appears when the target nucleic acid and a first nucleic acid and a second nucleic acid having a first flap site form a complex is cleaved with the first nucleic acid cleaving enzyme to generate a third nucleic acid, and the second flap site associated with a second invasion structure that appears when the third nucleic acid and a fourth nucleic acid having a second flap site form a complex on a fifth nucleic acid is cleaved with the second nucleic acid cleaving enzyme to generate a cleaved product.

[0011] The above problems are solved by any one of the following present inventions [1] to

[20] . [1] A nucleic acid detection method for detecting a target nucleic acid from a fluid containing the target nucleic acid, comprising a detection reagent mixing step of mixing a detection reagent comprising a first nucleic acid cleaving enzyme and a second nucleic acid cleaving enzyme into the fluid, wherein the first flap site associated with a first invasion structure that appears when the target nucleic acid and a first nucleic acid and a second nucleic acid having a first flap site form a complex is cleaved with the first nucleic acid cleaving enzyme to generate a third nucleic acid, and the second flap site associated with a second invasion structure that appears when the third nucleic acid and a fourth nucleic acid having a second flap site form a complex is cleaved with the second nucleic acid cleaving enzyme to generate a cleaved product. [2] The nucleic acid detection method according to [1], wherein the target nucleic acid is RNA, and at least the first flap region of the first nucleic acid, the second nucleic acid, and the fourth nucleic acid comprise DNA. [3] The nucleic acid detection method according to [1], wherein the target nucleic acid and the fourth nucleic acid are DNA, and the first nucleic acid and the second nucleic acid comprise RNA. [4] A nucleic acid detection method according to any one of [1] to [3], wherein the third nucleic acid, a fourth nucleic acid having a second flap site, and a fifth nucleic acid form a complex, thereby causing the second insertion structure to appear, and the fifth nucleic acid contains DNA. [5] A nucleic acid detection method described in any of [1] to [4], wherein the cleavage activity of the second nucleic acid cleaving enzyme on the first invasion structure is smaller than the cleavage activity of the first nucleic acid cleaving enzyme on the first invasion structure, and the cleavage activity of the first nucleic acid cleaving enzyme on the second invasion structure is smaller than the cleavage activity of the second nucleic acid cleaving enzyme on the second invasion structure. [6] The nucleic acid detection method described in [4], wherein the cleavage activity of the second nucleic acid cleaving enzyme on the first invasion structure is 90% or less of the cleavage activity of the first nucleic acid cleaving enzyme on the first invasion structure, and the cleavage activity of the first nucleic acid cleaving enzyme on the second invasion structure is 90% or less of the cleavage activity of the second nucleic acid cleaving enzyme on the second invasion structure. [7] A nucleic acid detection method described in any one of [1] to [6], wherein the cleavage activity of the first nucleic acid cleaving enzyme on the fourth nucleic acid is smaller than the cleavage activity of the second nucleic acid cleaving enzyme on the second invasive structure. [8] A nucleic acid detection method described in any of [1] to [7], wherein the cleavage activity of the first nucleic acid cleaving enzyme on the fourth nucleic acid and the cleavage activity of the second nucleic acid cleaving enzyme on the fourth nucleic acid are smaller than the cleavage activity of the first nucleic acid cleaving enzyme on the first invasion structure and the cleavage activity of the second nucleic acid cleaving enzyme on the second invasion structure. [9] The nucleic acid detection method described in [8], wherein the cleavage activity of the first nucleic acid cleaving enzyme on the fourth nucleic acid and the cleavage activity of the second nucleic acid cleaving enzyme on the fourth nucleic acid are 80% or less compared to the cleavage activity of the first nucleic acid cleaving enzyme on the first invasion structure and the cleavage activity of the second nucleic acid cleaving enzyme on the second invasion structure.

[10] The nucleic acid detection method according to any one of [1] to [9], wherein the concentration of the first nuclease is 9 times or less the concentration of the second nuclease.

[11] The nucleic acid detection method according to any one of [1] to

[10] , wherein the concentration of the first nuclease is 0.27 mg / mL or less.

[12] The nucleic acid detection method according to any one of [1] to

[11] , wherein the concentration of the second nucleic acid cleaving enzyme is 0.12 mg / mL or less.

[13] A nucleic acid detection method described in any of [1] to

[12] , further comprising a target nucleic acid confirmation step in which at least one of the first nucleic acid and the fourth nucleic acid that form the invasion structure is fluorescently labeled, and cleavage of the invasion structure can be detected by a change in fluorescence intensity.

[14] The nucleic acid detection method according to any one of [1] to

[12] , further comprising a target nucleic acid confirmation step of detecting the cleavage product by comparing the mobility before and after the reaction by electrophoresis.

[15] The nucleic acid detection method according to

[13] , wherein the production of the cleavage target by the first nucleic acid cleaving enzyme and the second nucleic acid cleaving enzyme in the detection reagent mixing step is carried out in a microspace.

[16] The nucleic acid detection method according to

[15] , wherein the height of the microscopic space is 10 nm to 100 μm.

[17] The microspace is 100,000 to 10 million particles / cm 2 The nucleic acid detection method according to

[15] or

[16] , wherein the electrodes are provided at a density of

[18] The nucleic acid detection method according to any one of

[15] to

[17] , wherein the target nucleic acid confirmation step detects the target nucleic acid by detecting the product to be cleaved produced in the microspace.

[19] The nucleic acid detection method according to any one of [1] to

[18] , wherein the first nuclease is a 5'-nuclease and the second nuclease is FEN-1.

[20] A detection reagent for carrying out the nucleic acid detection method according to any one of [1] to

[19] , comprising the first nucleic acid cleaving enzyme and the second nucleic acid cleaving enzyme. [Effects of the Invention]

[0012] According to the present invention, a nucleic acid detection method for rapidly detecting a target nucleic acid, such as RNA, from a fluid containing the target nucleic acid can be provided, and a detection reagent for carrying out the nucleic acid detection method can be provided. [Brief explanation of the drawings]

[0013] [Figure 1] 1 is a perspective view showing a microfluidic device according to an embodiment of the present invention. [Figure 2] FIG. 2 is a cross-sectional view taken along line II-II in FIG. [Figure 3A] FIG. 2 is a plan view showing a main part of a microwell array of the microfluidic device. [Figure 3B] 3B is a cross-sectional view taken along line IIIb-IIIb in FIG. 3A, showing a main part of a microwell array of the microfluidic device. FIG. [Figure 4] FIG. 2 is a diagram illustrating an example of use of the microfluidic device. [Figure 5] 1 is a captured image of a fluorescent field of view of the microfluidic device. [Figure 6] 1 is a graph showing the change over time in fluorescence intensity observed in an example. [Figure 7] 10 is a graph showing the change over time in fluorescence intensity observed in a comparative example. [Figure 8] 10 is a graph showing the change over time in fluorescence intensity observed in a comparative example. [Figure 9A] 1 is a graph showing the change over time in fluorescence intensity observed in Example 2. [Figure 9B] 1 is a graph showing the change over time in fluorescence intensity observed in Example 2. [Figure 9C] 1 is a graph showing the change over time in fluorescence intensity observed in Example 2. [Figure 9D] 1 is a graph showing the change over time in fluorescence intensity observed in Example 3. [Figure 9E] 1 is a graph showing the change over time in fluorescence intensity observed in Example 4. [Figure 9F] 1 is a graph showing the change over time in fluorescence intensity observed in Example 4. [Figure 10A] 1 shows photographed images of the fluorescent field (signal) of the microfluidic device observed in the examples. [Figure 10B] 1 is a photographed image of the fluorescent field (noise) of the microfluidic device observed in the example. [Figure 11]1 is a graph showing the change over time in fluorescence intensity observed in an example. [Figure 12] 1 is a graph showing the change over time in fluorescence intensity observed in an example. DETAILED DESCRIPTION OF THE INVENTION

[0014] [First embodiment] A nucleic acid detection method according to a first embodiment of the present invention will be described. The nucleic acid detection method according to this embodiment is a method for detecting a target nucleic acid from a fluid 20 containing the target nucleic acid.

[0015] The nucleic acid detection method according to this embodiment includes a detection reagent mixing step in which a detection reagent (microdroplets) 23 is mixed with a fluid 20.

[0016] The detection reagent 23 according to this embodiment includes a first nucleic acid cleaving enzyme and a second nucleic acid cleaving enzyme. The first nucleic acid cleaving enzyme and the second nucleic acid cleaving enzyme are enzymes that cleave specific flap sites associated with the invasion structure of a specific nucleic acid. To simplify the nucleic acid detection method according to this embodiment, the detection reagent 23 may contain, for example, a buffer and a surfactant. Typically, these reagents are provided in appropriate containers.

[0017] In this embodiment, the flap site refers to a site where, when nucleic acids are bound intramolecularly or intermolecularly, a part of the base sequence of the nucleic acid hybridizes by base pairing, but the other part at either the 3' end or the 5' end, or both, does not form base pairs. In this case, the part that does not form base pairs may consist of a normal nucleic acid base or a non-natural structure such as a fluorescent substance or an artificial nucleic acid base.

[0018] In the present embodiment, the nucleic acid invasion structure refers to a unique structure shown in Patent Documents 1 to 3 and Non-Patent Documents 1 and 2. That is, the nucleic acid invasion structure is a structure having a flap site that is generated when two types of oligonucleotide probes hybridize to a target nucleic acid to be detected and form a substrate complex.

[0019] The hybridizing oligonucleotide probe consists of a flap probe (FP) that has a base sequence complementary to the target nucleic acid and a base sequence that forms the flap site from the 5' end, and an invasion probe (IP) that hybridizes to a site adjacent to the hybridization position of the FP. At least one base at the 3' end of the IP has a base sequence that overlaps with the 5' portion where the FP hybridizes to the target nucleic acid. In the presence of an enzyme that recognizes a special structure such as an invasion structure and can cleave the flap site, the flap site is cleaved.

[0020] In the nucleic acid detection method according to this embodiment, after the detection reagent mixing step of mixing the fluid 20 with the detection reagent 23, a reaction for cleaving the invasive structure occurs in at least two stages.

[0021] In the first step, a first nucleic acid having a first flap site corresponds to the FP, and a second nucleic acid corresponds to the IP. The first nucleic acid and the second nucleic acid hybridize to a target nucleic acid to form a complex, thereby forming a first invasion structure. The first flap site associated with the first invasion structure is cleaved by a first nucleic acid cleaving enzyme, and a third nucleic acid is generated.

[0022] In the second step, the third nucleic acid generated in the first step corresponds to the IP in the second-step invasion structure, and the fourth nucleic acid, which has a second flap site and a base sequence to which the third nucleic acid can hybridize, corresponds to the FP, and these form a complex to form a second invasion structure. The second flap site associated with the second invasion structure is cleaved by a second nucleic acid cleaving enzyme to generate a cleavage target. In this case, the fourth nucleic acid has a base sequence to which the third nucleic acid can hybridize and has the combined function of the target nucleic acid in the first step and the FP, but the function may be divided by using the target nucleic acid in the second step as the fifth nucleic acid.

[0023] The cleavage target can be detected by comparing the mobility before and after the reaction by means of, for example, electrophoresis, etc. In addition, a fluorescent dye, a fluorescent substance having an absorption wavelength corresponding to the fluorescence wavelength of the fluorescent dye, and a quencher are attached to the first nucleic acid or the fourth nucleic acid, and the cleavage target can be identified from the luminescence that occurs when the two are separated by the cleavage reaction.

[0024] By confirming the cleavage target, it is possible to detect whether the target nucleic acid is contained in the fluid 20 (target nucleic acid confirmation step).

[0025] The nucleic acid detection method according to this embodiment is characterized in that the cleavage activity of the second nucleic acid cleaving enzyme on the first invasion structure is lower than that of the first nucleic acid cleaving enzyme on the first invasion structure, and the cleavage activity of the first nucleic acid cleaving enzyme on the second invasion structure is lower than that of the second nucleic acid cleaving enzyme on the second invasion structure. This characteristic is described in detail below.

[0026] Here, "cleavage activity" refers to the cleavage ability of an enzyme to recognize an invasive structure and cleave the flap portion, and can be expressed by detecting the amount of cleaved nucleic acid. In this embodiment, it can be expressed by comparing the mobility before and after the reaction by electrophoresis, or by measuring the fluorescence intensity due to the cleaved product. Cleavage activity can be adjusted by the enzyme concentration and reaction conditions. Furthermore, when the reaction conditions and enzyme concentration are constant, the cleavage activity increases hyperbolically toward a maximum value as the substrate concentration increases. When the substrate concentration is low, the substrate concentration and the cleavage activity are proportional. A low concentration is, for example, 1 mM or less, 10 μM or less, 10 nM or less, or 500 pM or less. Therefore, the enzyme concentration and the cleavage activity can be treated as being approximately equal.

[0027] The cleavage activity of the second nucleic acid cleaving enzyme against the first invasion structure is more than 0% and not more than 90% of that of the first nucleic acid cleaving enzyme against the first invasion structure, more preferably more than 0% and not more than 80%, even more preferably more than 0% and not more than 70%, and particularly preferably more than 0% and not more than 60%. On the other hand, the cleavage activity of the first nucleic acid cleaving enzyme against the second invasion structure is more than 0% and not more than 90% of the cleavage activity of the second nucleic acid cleaving enzyme against the second invasion structure, more preferably more than 0% and not more than 80%, even more preferably more than 0% and not more than 70%, and particularly preferably more than 0% and not more than 60%.

[0028] The first nuclease is an enzyme capable of recognizing the invasion structure and cleaving the flap site. The first nuclease has cleavage activity for the flap site associated with the DNA / DNA double-stranded structure formed when the target nucleic acid is DNA and the FP and IP are DNA. Furthermore, the first nuclease has cleavage activity against a flap site associated with an RNA / DNA double-stranded structure formed when the target nucleic acid is RNA and the FP and IP are DNA. For example, the first nucleic acid cleaving enzyme may be a polymerase-like nuclease, or may be any other nuclease, or may be a flap endonuclease modified to recognize an invasion structure in an RNA / DNA double-stranded structure.

[0029] The concentration of the first nucleic acid cleaving enzyme is preferably 9.0 times or less, more preferably 0.10 to 9.0 times, more preferably 0.6 to 2.5 times, and even more preferably 0.6 to 1.3 times the concentration of the second nucleic acid cleaving enzyme. When the concentration of the first nuclease is 9.0 times or less, the signal-to-noise ratio of detection is increased, facilitating nucleic acid detection. In particular, the background fluorescence intensity is suppressed while maintaining the fluorescence intensity in the second-step cleavage reaction. Here, the enzyme concentration refers to the concentration expressed in mg / mL.

[0030] The second nuclease is an enzyme capable of recognizing the invasion structure and cleaving the flap site. The second nuclease has higher cleavage activity than the first nuclease for the flap site associated with the DNA / DNA double-stranded structure formed when the target nucleic acid is DNA and the FP and IP are DNA. On the other hand, the second nucleic acid cleaving enzyme has a lower cleavage activity against the flap site associated with the RNA / DNA double-stranded structure formed when the target nucleic acid is RNA and the FP and IP are DNA (e.g., the first invasion structure with RNA as the target nucleic acid) than the first nucleic acid cleaving enzyme. The activity rate is preferably lower than 100%, more preferably lower than 90%, more preferably lower than 80%, more preferably lower than 70%, more preferably lower than 60%, more preferably lower than 50%, more preferably lower than 40%, more preferably lower than 30%, more preferably lower than 20%, and more preferably lower than 10%. For example, the second nucleic acid-cleaving enzyme may be a flap endonuclease, or may be any other nuclease, or may be a polymerase nuclease modified to recognize an invasion structure in a DNA / DNA double-stranded structure.

[0031] The concentration of the second nucleic acid cleaving enzyme is preferably 0.1 times or more, more preferably 0.1 to 10 times, more preferably 0.4 to 1.7 times, and even more preferably 0.75 to 1.7 times the concentration of the second nucleic acid cleaving enzyme. The concentration of the second nucleic acid cleaving enzyme is preferably 0.12 mg / mL or less, more preferably 0.008 to 0.12 mg / mL, and even more preferably 0.03 to 0.06 mg / mL. When the concentration of the second nucleic acid cleaving enzyme is 0.1 times or more the concentration of the first nucleic acid cleaving enzyme, the reaction time required for detection can be shortened while noise can be reduced. Furthermore, when the concentration of the second nucleic acid cleaving enzyme is 0.12 mg / mL or less, the reaction time can be shortened while noise can be reduced.

[0032] The nucleic acid detection method according to the present embodiment is highly effective in detecting the presence or absence of a target nucleic acid by recognizing an invasive structure and causing a cleavage reaction. For example, by mixing enzymes with different activities against the nucleic acid structure to be cleaved in the same reaction system, the detection signal is enhanced and noise is reduced, thereby increasing the signal-to-noise ratio during detection.

[0033] In conventional nucleic acid detection methods, nucleic acid cleavage reactions using enzymes can sometimes result in undesired side reactions due to misrecognition of certain nucleic acid structures. For example, in the target nucleic acid confirmation step described above, a reaction can occur with only the reagent, resulting in fluorescence emission, even when the target nucleic acid is not present. Specifically, when the target nucleic acid is RNA, a detection system using a single type of nucleic acid cleavage enzyme, particularly a first nucleic acid cleavage enzyme, is prone to a side reaction in which the flap site of the fourth nucleic acid is cleaved. Therefore, if the amount of enzyme input into the reaction system is increased to promote the cleavage reaction of the flap site in order to shorten the detection time, the aforementioned side reaction will increase. If the amount of first nucleic acid cleavage enzyme input is reduced to avoid this, the detection time will increase.

[0034] The above-mentioned side reactions can occur similarly even when two types of nucleic acid cleaving enzymes are used. In the nucleic acid detection method according to this embodiment, it is possible to suppress the above-mentioned side effects and increase the signal-to-noise ratio during detection without increasing the detection time. The amount of the first nucleic acid cleaving enzyme in the detection reagent 23 is reduced to suppress the above-mentioned side reactions. Furthermore, by mixing a second nucleic acid cleaving enzyme into the detection reagent 23, the cleavage activity of the second-stage cleavage reaction (cleavage reaction on the second invasive structure consisting of the third nucleic acid and the fourth nucleic acid) is enhanced. By reducing the amount of the first nucleic acid cleaving enzyme, side reactions are suppressed, while by mixing the second nucleic acid cleaving enzyme, an increase in detection time is prevented.

[0035] The cleavage activity of the first nuclease on the fourth nucleic acid is lower than the cleavage activity of the second nuclease on the second invasion structure.

[0036] The cleavage activity of the first nucleic acid cleaving enzyme on the fourth nucleic acid and the cleavage activity of the second nucleic acid cleaving enzyme on the fourth nucleic acid are smaller than the cleavage activity of the first nucleic acid cleaving enzyme on the first invasion structure and the cleavage activity of the second nucleic acid cleaving enzyme on the second invasion structure.

[0037] The cleavage activity of the first nucleic acid cleaving enzyme on the fourth nucleic acid and the cleavage activity of the second nucleic acid cleaving enzyme on the fourth nucleic acid are 80% or less, preferably 60% or less, more preferably 50% or less, more preferably 40% or less, and even more preferably 30% or less, of the cleavage activity of the first nucleic acid cleaving enzyme on the first invasion structure and the cleavage activity of the second nucleic acid cleaving enzyme on the second invasion structure.

[0038] The concentration of the first nuclease is 9.0 times or less, more preferably 0.1 to 9.0 times, more preferably 0.6 to 2.5 times, and even more preferably 0.6 to 1.3 times the concentration of the second nuclease.

[0039] The concentration of the first nuclease is 0.27 mg / mL or less, more preferably 0.18 mg / mL or less, more preferably 0.148 mg / mL or less, more preferably 0.09 mg / mL or less, more preferably 0.074 mg / mL or less, more preferably 0.045 mg / mL or less, and even more preferably 0.037 mg / mL or less. Also, the concentration of the first nuclease is 0.003 mg / mL or more, more preferably 0.005 mg / mL or more, more preferably 0.01 mg / mL or more, and even more preferably 0.019 mg / mL or more.

[0040] Here, the second nucleic acid cleaving enzyme has a lower cleavage activity against the flap site associated with an RNA / DNA double-stranded structure (e.g., a first invasion structure with RNA as the target nucleic acid) than the first nucleic acid cleaving enzyme. Therefore, even if the second nucleic acid cleaving enzyme is further mixed into the detection reagent 23, it has little effect on the cleavage activity of the first-stage cleavage reaction by the first nucleic acid cleaving enzyme (the cleavage reaction against the first invasion structure consisting of the target nucleic acid, the first nucleic acid, and the second nucleic acid).

[0041] Furthermore, according to the nucleic acid detection method of this embodiment, the amount of the first nucleic acid cleaving enzyme can be reduced, and the first cleaving enzyme can be prevented from cleaving the second invasive structure, thereby increasing the signal-to-noise ratio during detection and enabling more effective detection of the target nucleic acid.

[0042] The formation of a double-stranded structure can be carried out under normal conditions such as temperature, pH, salt concentration, and buffer solution, but is preferably carried out in the same solution as the solution in which the nucleic acid cleavage reaction is carried out. Hybridization is preferably carried out in a reaction solution containing a buffering salt. The pH of the reaction solution is preferably in the range of 6.5 to 10.0, particularly 7.5 to 9.0. The concentration of the buffering salt is preferably in the range of 5 to 250 mM, particularly 10 to 100 mM. Examples of buffering salts include cacodylate, phosphate, and Tris salt, but other salts may also be used.

[0043] The reaction solution preferably contains a salt of an alkali metal and / or alkaline earth metal, such as sodium chloride and / or magnesium chloride. The salt concentration in the reaction solution is preferably in the range of 1 to 100 mM, more preferably in the range of 5 to 50 mM, and even more preferably in the range of 10 to 30 mM.

[0044] The temperature throughout the reaction is preferably in the range of 15 to 90°C, particularly preferably in the range of 25 to 80°C, and particularly preferably in the range of 35 to 70°C.

[0045] The nucleic acid to be detected in the nucleic acid detection method according to this embodiment is not particularly limited as long as it is DNA or RNA, and may be natural or synthetic. Examples of natural nucleic acids include genomic DNA, mRNA, rRNA, hnRNA, miRNA, and tRNA recovered from living organisms. Synthetic nucleic acids include DNA synthesized by known chemical synthesis methods such as the β-cyanoethyl phosphoramidite method and DNA solid-phase synthesis, nucleic acids synthesized by known nucleic acid synthesis methods such as PCR, and cDNA synthesized by reverse transcription.

[0046] The nucleic acid to be detected in the nucleic acid detection method according to this embodiment is not particularly limited, and may be nucleic acid extracted from an organism such as an animal, a plant, a microorganism, or a cultured cell, or may be nucleic acid amplified by a nucleic acid amplification reaction. When the amplification product is a double-stranded nucleic acid, it can be used after being converted into single strands by thermal denaturation, chemical denaturation, or the like. Examples of such nucleic acid amplification reactions include PCR, LAMP, SMAP, NASBA, and RCA. Nucleic acids can also be extracted from animals and the like by known techniques such as the phenol / chloroform method.

[0047] (Effects of the first embodiment) According to the nucleic acid detection method of this embodiment, the signal-to-noise ratio during detection can be increased without increasing the detection time, which is particularly effective when RNA is used as the target nucleic acid. Furthermore, according to the nucleic acid detection method of this embodiment, nucleic acid can be easily detected by simply mixing the detection reagent 23, which is a minute droplet, with the fluid 20 just once. The ease and speed of RNA detection will enable testing to be performed in hospitals on the day of consultation. This will allow for quick decisions on treatment and medication dosage, preventing patients from becoming seriously ill. It is also expected that reducing the number of visits to the hospital will contribute to reducing medical manpower shortages and medical costs.

[0048] [Second embodiment] A nucleic acid detection method according to a second embodiment of the present invention will be described with reference to Figures 1 to 3. Note that the dimensions of each component have been adjusted appropriately to make the drawings easier to see. In the following description, components that are common to those already described will be assigned the same reference numerals, and duplicated descriptions will be omitted.

[0049] The nucleic acid detection method according to the second embodiment is carried out using a vessel in which tubes or wells found in conventional biochemical or molecular biological techniques are arranged on a plate. In addition, microwell arrays with various types of minute channel structures formed using etching techniques, photolithography, and other techniques used in semiconductor circuit manufacturing have been studied in recent years. The wells of these microwell arrays can also be used as chemical reaction vessels for carrying out various biochemical or chemical reactions in minute volumes of fluid. An infinite number of microwells exist on a single array, with each microwell containing one target substance. By making it possible to confirm the presence or absence of the target substance by fluorescence or other means, the target substance can be quantified by counting the number of wells in which fluorescence is observed. The nucleic acid detection method according to the second embodiment uses a microwell array as a chemical reaction vessel.

[0050] First, a microfluidic device 1 used in a digital counting method using liquid biopsy will be described as an analytical device used in the nucleic acid detection method according to this embodiment. FIG. 1 is a perspective view showing a microfluidic device 1. As shown in FIG. FIG. 2 is a cross-sectional view taken along line II-II in FIG. FIG. 3 is a diagram showing the main part of the microwell array 4 of the microfluidic device 1. As shown in FIG. 3A is a plan view thereof, and FIG. 3B is a cross-sectional view taken along line IIIb-IIIb in FIG. 3A. The microfluidic device 1 can be used not only for liquid biopsy but also for measuring proteins, DNA, RNA, viral biomolecules, bacteria, etc.

[0051] As shown in FIGS. 1 to 3, a microfluidic device 1 includes a microwell array 4 in which a plurality of wells (microspaces) 3 are formed. As shown in Figures 1 and 2, the microwell array 4 comprises a plate-shaped bottom layer 11 and a plate-shaped wall layer 12 formed on the bottom layer 11. The multiple wells 3 of the microwell array 4 are arranged in an array, as shown in Figure 3A. In Figure 3A, the wells 3 are arranged at intervals in the horizontal direction (left and right direction in Figure 3A). Furthermore, multiple horizontally arranged well groups 30, each consisting of multiple horizontally arranged wells 3, are arranged in the vertical direction (up and down direction in Figure 3A). Vertically adjacent horizontally arranged well groups 30 are arranged with a horizontal offset so that the wells 3 do not overlap each other in the vertical direction.

[0052] The surface (opening surface 6) of the microwell array 4 onto which the multiple wells 3 are open is flat. The multiple wells 3 may be formed, for example, over the entire opening surface 6 of the microwell array 4, but in this embodiment, they are formed in a region of the opening surface 6 of the microwell array 4 excluding the peripheral region 7 (well formation region 8). The peripheral shape of the well formation region 8 may be any shape, but in this embodiment, it is rectangular.

[0053] As shown in Figures 2 and 3, the wells 3 are holes with a bottom. In this embodiment, the wells 3 are formed in a cylindrical shape and have a bottom surface 9. There are no particular limitations on the shape or size of the wells 3 of the microwell array 4, but when various biochemical reactions are carried out in microdroplets, for example, it is preferable that the wells have a shape and size that can accommodate one to several biomolecules 21 or carriers. The carriers may be, for example, beads, and the biomolecules 21 that are samples may be bound to the carriers.

[0054] The shape of the well 3 is not limited to a cylindrical shape and may be any shape, such as a cylindrical shape, a polyhedron formed with multiple faces (e.g., a rectangular parallelepiped, a hexagonal prism, an octagonal prism, etc.), an inverted cone shape, or an inverted pyramid shape (e.g., an inverted triangular pyramid, an inverted square pyramid, an inverted pentagonal pyramid, an inverted hexagonal pyramid, or an inverted polygonal pyramid with seven or more sides). Here, the inverted cone shape and the inverted pyramid shape refer to the shape of the well 3 in which the bottom of the cone and the pyramid, respectively, form the opening of the well 3. When the shape of the well 3 is an inverted cone shape or an inverted pyramid shape, the apex of the cone or the pyramid may be cut off to make the bottom surface 9 of the well 3 flat. As another example, the bottom surface 9 of the well 3 may be formed into a curved surface that is convex or concave toward the opening of the well 3. The shape of the well 3 may also be a combination of two or more of the above shapes.

[0055] For example, the shape of the well 3 may be partially cylindrical and the remaining portion in the shape of an inverted cone. In the case of an inverted cone or pyramid, the bottom of the cone or pyramid serves as the entrance (opening) of the well 3, but the well 3 may also have a shape in which a portion of the top of the inverted cone or pyramid is cut off. In this case, the bottom of the microwell will be flat.

[0056] 3B, in this embodiment, the well 3 has a cylindrical shape, and the bottom of the well 3 is flat, but may also be curved (convex or concave). The bottom of the well 3 can also be curved when the well 3 has a shape obtained by cutting off a portion of the apex of an inverted cone or inverted pyramid.

[0057] 3B, when well 3 is cylindrical, the maximum diameter and height Wh of well 3 may be, for example, 10 nm to 100 μm, preferably 100 nm to 20 μm, and more preferably 1 μm to 20 μm, for the purpose of enclosing an aqueous liquid containing biomolecules 21. The dimensions of well 3 are determined appropriately, taking into consideration the amount of aqueous liquid to be contained in well 3 and the preferred ratio between the size of carriers such as beads to which biomolecules 21 are attached and the dimensions of well 3, so that one or several biomolecules 21 can be contained in one microwell.

[0058] The material of the microwell array 4 is preferably one that does not inhibit the enzyme reaction. Alternatively, the surface of the material of the microwell array 4 can be coated with a substance that does not inhibit the enzyme reaction. For example, a surfactant, a phospholipid, another polymer compound, or a mixture of these may be coated. Examples of surfactants include nonionic surfactants. Examples of nonionic surfactants include Tween, glycerol, and Triton-X100. Examples of polymer compounds include polyethyleneglycol (PEG), DNA, proteins, and BSA. The surface of the material of the microwell array 4 may also be coated with a fluorine-based coating agent or a silicone-based coating agent. By coating with these coating agents, the surface can be modified to be hydrophobic. As a result of coating in this manner, it is possible to prevent aqueous solutions, buffers, reagents, enzymes, nucleic acids, and other substances contained in the reaction system from adhering to the surface, thereby preventing false detection.

[0059] The detection target in the nucleic acid detection method using microwell array 4 according to this embodiment may be, for example, a sample collected from a living body such as blood, a PCR product, etc., or an artificially synthesized compound, etc. For example, when DNA, which is a biomolecule 21, is the detection target, well 3 may have a shape and size that allows one DNA molecule to be placed inside.

[0060] The microwell array 4 has wells 3 with a density of, for example, 100,000 to 10,000,000 / cm 2 and preferably 100,000 to 5,000,000 particles / cm 2 and more preferably 100,000 to 1,000,000 particles / cm 2 When the density of the wells 3 is within this range, it is easy to seal the aqueous liquid sample into a predetermined number of wells 3. In addition, it is also easy to observe the wells 3 to analyze the experimental results.

[0061] For example, when measuring mutations in cell-free DNA, if the proportion of the mutation to be detected relative to the wild type is about 0.01%, it is suitable to use, for example, 1 to 2 million wells.

[0062] In the microwell array 4, each well 3 can be fabricated by a process of forming a hydrophilic or hydrophobic layer by layering a hydrophilic or hydrophobic material on a substrate, and then excavating the hydrophilic or hydrophobic layer to form multiple wells. However, as long as the conditions allow for realization of this embodiment, the wells 3 may be formed by molding or cutting using the same material as the substrate.

[0063] The microwell array 4 may be formed on a substrate. The substrate may or may not be electromagnetically transparent. Examples of electromagnetic waves include X-rays, ultraviolet light, visible light, and infrared light. When the microwell array is formed on an electromagnetically transparent substrate, electromagnetic waves can be used to analyze the results of experiments performed on the microwell array. For example, fluorescence, phosphorescence, and the like resulting from irradiation with electromagnetic waves can be measured from the substrate side. Fluorescence, phosphorescence, and the like can be detected using, for example, a fluorescence microscope. In this case, electromagnetic waves may be irradiated from, for example, the substrate side, or from, for example, the entrance side of the wells.

[0064] For example, when detecting fluorescence having a peak in the wavelength range of 400 to 700 nm, which is the visible light region, in microwell array 4, a substrate having good transparency to at least light in the visible light region may be used.

[0065] Examples of substrates that are electromagnetically transparent include glass and resin. Examples of resin substrates include ABS resin, polycarbonate resin, COC (cycloolefin copolymer), COP (cycloolefin polymer), acrylic resin, polyvinyl chloride, polystyrene resin, polyethylene resin, polypropylene resin, polyvinyl acetate, PET (polyethylene terephthalate), PEN (polyethylene naphthalate), etc. These resins may contain various additives, or multiple resins may be mixed.

[0066] From the viewpoint of using fluorescence or phosphorescence to detect experimental results, the substrate preferably has substantially no autofluorescence. Here, "substantially no autofluorescence" means that the substrate has no autofluorescence at the wavelength used to detect experimental results, or if it does have autofluorescence, it is so weak that it does not affect the detection of the experimental results. For example, if the intensity of the autofluorescence is about 1 / 2 or less or 1 / 10 or less of the fluorescence to be detected, it can be said to be so weak that it does not affect the detection of the experimental results.

[0067] Examples of materials that are electromagnetically transparent and emit no autofluorescence include quartz glass. Materials that emit weak autofluorescence and do not interfere with the detection of experimental results using electromagnetic waves include low-fluorescence glass, acrylic resin, COC (cycloolefin copolymer), COP (cycloolefin polymer), etc.

[0068] The microwell array 4 may be one in which a plurality of wells 3 are formed only by resin molding using injection molding or imprinting.

[0069] Fluorescence used to detect biomolecules 21 includes those derived from fluorescent molecules that can label nucleic acids, fluorescent beads that encapsulate fluorescent molecules, and intercalators such as SYBR Green that specifically enter the double helix of DNA and emit fluorescence.

[0070] The fluorescence emitted from the biomolecules 21 contained in the wells 3 can be observed from the substrate side of the microwell array 4 using, for example, a fluorescence microscope. By observing the fluorescence, the number of wells 3 that emit a predetermined fluorescence can be counted, thereby identifying the number of target molecules.

[0071] For example, the biomolecule 21 may be placed in the well 3 as is, or may be treated with a fluorescent label that specifically labels the target molecule before being placed in the well 3. Alternatively, the target molecule may be captured using beads that specifically recognize the target molecule, and then the beads may be placed in the well 3 and brought into contact with a fluorescent label that can specifically label the target molecule, thereby fluorescently labeling the target molecule in the well 3.

[0072] The thickness of the substrate can be determined as appropriate, but when observing fluorescence from the substrate side using a fluorescence microscope, it is preferable that the thickness be, for example, more than 0 mm and not more than 5 mm, more than 0 mm and not more than 2 mm, or more than 0 mm and not more than 0.6 mm.

[0073] In addition to fluorescence, for example, turbidity can also be used to observe biomolecules 21 using microwell array 4. Turbidity can be measured by the transmittance of light with a wavelength of, for example, about 400 to 1000 nm.

[0074] The resin forming the hydrophilic portion (hereinafter sometimes referred to as "hydrophilic resin") is not particularly limited as long as it exhibits the effects of the present invention, but examples thereof include resins in which the molecules of the constituent components of the resin have hydrophilic groups that exhibit hydrophilicity. Examples of hydrophilic groups include hydroxyl groups, carboxyl groups, sulfone groups, sulfonyl groups, amino groups, amide groups, ether groups, and ester groups.

[0075] More specifically, the hydrophilic resin may be appropriately selected from siloxane polymers, epoxy resins, polyethylene resins, polyester resins, polyurethane resins, polyacrylamide resins, polyvinylpyrrolidone resins, acrylic resins such as polyacrylic acid copolymers, polyvinyl alcohol resins such as cationized polyvinyl alcohol, silanolated polyvinyl alcohol, and sulfonated polyvinyl alcohol, polyvinyl acetal resins, polyvinyl butyral resins, polyethylene polyamide resins, polyamide polyamine resins, cellulose derivatives such as hydroxymethyl cellulose and methyl cellulose, polyalkylene oxide derivatives such as polyethylene oxide and polyethylene oxide-polypropylene oxide copolymers, maleic anhydride copolymers, ethylene-vinyl acetate copolymers, styrene-butadiene copolymers, and combinations of the above resins. The hydrophilic resin may be a thermoplastic resin, a thermosetting resin, a resin that cures with active energy rays such as electron beams or UV light, or an elastomer.

[0076] The hydrophilic portion (material of the hydrophilic portion) may have a contact angle of less than 70 degrees measured according to the sessile drop method specified in JIS R3257-1999.

[0077] In addition, when the substrate described above is present, the hydrophilic layer may also have the function of adhering the substrate and the hydrophobic layer to each other. For example, the hydrophilic layer may be formed by applying a thermosetting silane coupling agent or the like to the substrate and thermally curing the agent to form a siloxane polymer.

[0078] The resin forming the hydrophobic portion (hereinafter sometimes referred to as "hydrophobic resin") is not particularly limited as long as it exhibits the effects of the present invention, but may be appropriately selected from, for example, novolak resins, acrylic resins, methacrylic resins, styrene resins, vinyl chloride resins, vinylidene chloride resins, polyolefin resins, polyamide resins, polyimide resins, polyacetal resins, polycarbonate resins, polyphenylene sulfide resins, polysulfone resins, fluororesins, silicone resins, urea resins, melamine resins, guanamine resins, phenolic resins, cellulose resins, and combinations of the above resins, and may be used if the contact angle measured in accordance with the sessile drop method specified in JIS R3257-1999 is 70 degrees or more. The hydrophobic resin may be a thermoplastic resin, a thermosetting resin, a resin that cures with active energy rays such as electron beams or UV light, or an elastomer.

[0079] The hydrophobic portion may be formed of, for example, a resist. The resist may be a photoresist, which facilitates the formation of a fine structure. The photoresist may be, for example, a photosensitive novolac resin.

[0080] As shown in Figures 1 and 2, microwell array 4 is provided with a lid member 5 which, together with microwell array 4, constitutes flow path 2 of microfluidic device 1. Lid member 5 is a plate- or sheet-shaped member, and is disposed opposite opening surface 6 of microwell array 4. Between microwell array 4 and lid member 5, there is a gap which functions as flow path 2.

[0081] The cover member 5 has a plurality of through-holes 15 penetrating in its thickness direction. Each through-hole 15 communicates with the flow channel 2 of the microfluidic device 1 and functions as an inlet for supplying fluid into the flow channel 2 and an outlet for discharging the fluid. In this embodiment, the cover member 5 has two through-holes 15A, 15B (a first through-hole 15A and a second through-hole 15B). The two through-holes 15A, 15B are arranged at positions corresponding to two opposing corners of the rectangular well formation region 8. Each through-hole 15 has a small hole portion 16 and a large hole portion 17 that is larger than the small hole portion 16 when viewed from the penetration direction.

[0082] (Effects of the second embodiment) According to the nucleic acid detection method of this embodiment, similarly to the first embodiment, it is possible to increase the signal-to-noise ratio during detection without increasing the detection time by simply mixing the detection reagent 23 with the fluid 20 once. Furthermore, the detection results can be effectively visualized using the well (microspace) 3.

[0083] Next, a nucleic acid detection method using the microfluidic device 1 having the microwell array 4 according to this embodiment will be described. Figures 4 and 5 are diagrams illustrating an example of how the microfluidic device 1 is used.

[0084] First, a fluid 20 containing a biomolecule 21, which is a target nucleic acid, and a sealing liquid are prepared. The fluid 20 is composed of the biomolecule 21 and a solvent fluid 22, and is further mixed with a detection reagent 23. The sealing liquid is, for example, oil.

[0085] Next, the microfluidic device 1 is placed so that the flow of the fluid 20 and the sealing liquid in the flow channel 2 is horizontal, and the opening of the well 3 faces upward in the vertical direction. Next, a pipette is used to inject the fluid 20 into the channel 2 through the first through-hole 15A of the microfluidic device 1. Figure 4 shows the state after the injection of the fluid 20 has been completed.

[0086] Thereafter, a sealing liquid is injected into the flow channel 2 through the first through-hole 15A of the microfluidic device 1, filling the flow channel 2 with the sealing liquid. In other words, the fluid 20 in the flow channel 2 is replaced with the sealing liquid. As a result, the fluid 20 in each well 3 is sealed with the sealing liquid. After sealing, the microfluidic device 1 is heated, causing a biochemical reaction inside. If the fluid 20 contains a target nucleic acid, a cleavage target is generated by a cleavage reaction in the two-stage penetration structure. By detecting the cleavage target, it is possible to detect that a biomolecule 21 is stored in the well 3.

[0087] Signals to be measured in the wells 3 of the microwell array 4 after the reaction include fluorescence, luminescence, phosphorescence, etc. These wavelength ranges can be those observable as electromagnetic waves, such as visible light, ultraviolet light, and infrared light. Therefore, a detection device having a measurement device capable of measuring such wavelength ranges can be used as the detector according to this embodiment. Specifically, in this embodiment, a fluorescence microscope is used as a detector for detecting the reaction.

[0088] When the microwell array 4 is configured to transmit light, the microfluidic device 1 is positioned so that it can be observed from the microwell array 4 side with a fluorescence microscope. An image observed with a fluorescence microscope is shown, for example, in Figure 5. In the example of Figure 5, fluorescence is observed with a fluorescence microscope.

[0089] The present invention will be further described below with reference to examples, but the present invention is not limited to these examples. [Example]

[0090] In the following Examples and Comparative Examples, RNA detection was carried out by Invasive Cleavage Assay using the sequences shown in Table 1 under the following reagent amounts and conditions. The underlined portion of the first nucleic acid in Table 1 represents the first flap site. The underlined portion of the fourth nucleic acid represents the second flap site. In addition, F in the fourth nucleic acid represents a fluorescent dye, and Q represents a quencher.

[0091] In an embodiment, the first nucleic acid and the second nucleic acid hybridize on the target RNA, and the first nucleic acid cleaving enzyme cleaves the first flap site to generate a third nucleic acid. The third nucleic acid is hybridized to the fourth nucleic acid, and the second flap site is cleaved by the second nucleic acid cleaving enzyme, resulting in the release of the third nucleic acid. This separates the fluorescent substance from the quencher, allowing fluorescence emission to be observed.

[0092] [Table 1]

[0093] Example 1 In this example, nucleic acid detection was carried out using a detection reagent containing a first nuclease and a second nuclease.

[0094] (conditions) A reaction mixture containing target RNA (30 pM, 0 pM), first nucleic acid (1 μM), second nucleic acid (1 μM), fourth nucleic acid (2 μM), 3-morpholinopropanesulfonic acid (pH 7.9, 10 mM), MgCl2 (10 mM), Tween 20 (0.05% v / v), 5'-nuclease (molecular weight: 92290) (0.09 mg / mL) as the first nuclease, Flapendo Nuclease 1 (molecular weight: 36960) (0.03 mg / mL) as the second nuclease, and distilled water was prepared in a total volume of 10 μL per reaction. Two reaction mixtures with different concentrations of target RNA were prepared.

[0095] (procedure) The reaction solution was placed in a micro test tube and heated at 65°C for 60 minutes in a real-time PCR device, and the change in fluorescence intensity (excitation: 490 nm, emission: 520 nm) was measured.

[0096] (result) The results are shown in Figure 6. It was observed that the higher the concentration of target RNA, the faster the rise in fluorescence intensity.

[0097] Comparative Example 1 In this example, nucleic acid detection was carried out using a detection reagent containing the second nuclease but not the first nuclease.

[0098] (conditions) The conditions were the same as in Example 1, except that the first nuclease was not contained and Flapendo Nuclease 1 (0.03 mg / mL) was mixed as the second nuclease.

[0099] (procedure) The procedure is the same as in Example 1.

[0100] (result) The results are shown in Figure 7. No change in fluorescence intensity was observed depending on the concentration of target RNA.

[0101] Comparative Example 2 In this example, nucleic acid detection was carried out using a detection reagent containing a first nuclease but not a second nuclease.

[0102] (conditions) The conditions were the same as in Example 1, except that the second nuclease was not contained and 5'-nuclease (0.09 mg / mL) was mixed as the first nuclease.

[0103] (procedure) The procedure is the same as in Example 1.

[0104] (result) The results are shown in Figure 8. It was observed that the higher the concentration of target RNA (30 pM), the faster the rise in fluorescence intensity.

[0105] In Example 1, the rise in fluorescence intensity is faster than when one type of nucleic acid cleaving enzyme is used (Comparative Examples 1 and 2) under the same target RNA concentration conditions. The results of Example 1 show that when RNA is used as the target nucleic acid, the signal-to-noise ratio of detection can be improved by using detection reagent 23 containing the first nuclease and the second nuclease. Furthermore, it has been shown that the use of detection reagent 23 containing the first nuclease and the second nuclease accelerates the rise of fluorescence intensity, preventing an increase in detection time.

[0106] Example 2 In this example, the effect of the activity ratio between the first nuclease and the second nuclease on the signal-to-noise ratio in the detection of target RNA was confirmed.

[0107] (conditions) The concentration ratios of the first nuclease cleaving enzyme 5'-nuclease contained in the reaction solution were 0.10 eq. (0.003 mg / mL), 0.17 eq. (0.005 mg / mL), 0.33 eq. (0.010 mg / mL), 0.63 eq. (0.019 mg / mL), 1.23 eq. (0.037 mg / mL), 1.5 eq. (0.045 mg / mL), 2.46 eq. (0.074 mg / mL), and 3.0 e The conditions were the same as in Example 1, except that 13 different reaction solutions were used, varying the concentrations of the target RNA: q. (0.09 mg / mL), 4.93 eq. (0.148 mg / mL), 6.0 eq. (0.18 mg / mL), 9.0 eq. (0.27 mg / mL), 12.0 eq. (0.36 mg / mL), and 15.0 eq. (0.45 mg / mL), and that the target RNA concentration was 30 pM and / or 300 pM. The second nucleic acid cleaving enzyme was Flapendo Nuclease 1 (0.03 mg / mL), as in Example 1.

[0108] (procedure) The procedure is the same as in Example 1.

[0109] (result) The results are shown in Figures 9A to 9C. When the target RNA concentrations were 30 pM and 300 pM, no change in the rise of the signal due to the concentration ratio of the first nuclease was observed. When the target RNA concentration was 0 pM, it was confirmed that the rise became faster as the concentration ratio of the first nuclease increased. This is because the small amount of the first nuclease suppressed side reactions and reduced noise. Furthermore, the addition of the second nuclease prevented an increase in detection time. Furthermore, by adding a second nuclease that does not have high activity against the first invasive structure, the detection reaction is maintained and the signal-to-noise ratio (the difference in fluorescence intensity between when the target RNA concentration is 30 pM or 300 pM and when it is 0 pM) is increased. In particular, it has been shown that a favorable signal-to-noise ratio is obtained when the concentration ratio of the first nucleoside cleaving enzyme 5'-nuclease is 0.10x, 0.17x, 0.33x, 0.63x, 1.23x, 1.5x, 2.46x, 3.0x, 4.93x, 6.0x, or 9.0x, and that an even more favorable signal-to-noise ratio is obtained when the concentration ratio of the first nucleoside cleaving enzyme 5'-nuclease is 0.10x, 0.17x, 0.33x, 0.63x, 1.23x, 1.5x, 2.46x, 3.0x, 4.93x, or 6.0x.

[0110] Example 3 In this example, the effect of the activity ratio between the first nuclease and the second nuclease on the signal-to-noise ratio in target RNA detection was confirmed, regardless of the concentration of the second nuclease.

[0111] (conditions) The conditions were the same as in Example 1, except that three types of reaction solutions were used in which the concentration ratio of the first nuclease 5'-nuclease contained in the reaction solution was changed to 0.63 eq. (0.037 mg / mL), 1.23 eq. (0.074 mg / mL), and 2.46 eq. (0.148 mg / mL), the target RNA concentration was 30 pM and / or 300 pM, and the second nuclease Flapendo Nuclease 1 was 0.06 mg / mL.

[0112] (procedure) The procedure is the same as in Example 1.

[0113] (result) The results are shown in Figure 9D. When the target RNA concentration was 30 pM and 300 pM, no change in the rise of the signal was observed depending on the concentration ratio of the first nuclease. When the target RNA concentration was 0 pM, it was confirmed that the rise became faster as the concentration ratio of the first nuclease increased. This is because the small amount of the first nuclease inhibited side reactions and reduced noise. Furthermore, the addition of the second nuclease prevented an increase in detection time. Furthermore, the addition of a second nuclease with low activity against the first invasive structure maintained the detection reaction and increased the signal-to-noise ratio (the difference in fluorescence intensity between a target RNA concentration of 30 pM or 300 pM and a target RNA concentration of 0 pM). These results, along with those of Example 2, demonstrate that a favorable signal-to-noise ratio is achieved regardless of the concentration of the second nuclease. In particular, favorable signal-to-noise ratios were achieved when the concentration ratio of the first nuclease 5'-nuclease was 0.63, 1.23, and 2.46. Furthermore, it was shown that when the concentration ratio of the first nuclease 5'-nuclease was 0.63 or 1.23, a suitable signal-to-noise ratio was obtained regardless of the concentration of the target RNA. Thus, when the concentration ratio of the first nuclease 5'-nuclease was 0.6 to 1.3, target nucleic acids of various concentrations could be detected with a suitable signal-to-noise ratio, and a concentration of the second nuclease of 0.03 mg / mL to 0.06 mg / mL was even more suitable.

[0114] Example 4 In this example, the effect of the activity ratio between the first nuclease and the second nuclease on the signal-to-noise ratio in target RNA detection was confirmed. In this example, the concentration of the first nuclease was fixed at 0.63 eq., and experiments were performed by varying the concentration of the second nuclease.

[0115] (conditions) The conditions were the same as in Example 1, except that five types of reaction solutions were required: the first nuclease 5'-nuclease had a concentration ratio of 0.63 eq. in the reaction solution; the second nuclease Flapendo Nuclease 1 was at 0.008 mg / mL, 0.015 mg / mL, 0.06 mg / mL, 0.12 mg / mL, and 0.24 mg / mL; and the first nuclease 5'-nuclease was at 0.005 mg / mL, 0.01 mg / mL, 0.037 mg / mL, 0.074 mg / mL, and 0.148 mg / mL to match the concentrations of the second nuclease; and the target RNA concentration was 30 pM and / or 300 pM.

[0116] (procedure) The procedure is the same as in Example 1.

[0117] (result) The results are shown in Figures 9E and 9F. When the target RNA concentrations were 30 pM and 300 pM, no change in the signal rise was observed depending on the concentration of the second nuclease. When the target RNA concentration was 0 pM, it was confirmed that the rise became faster as the concentration of the second nuclease increased, but no significant difference was observed at second nuclease concentrations of 0.06 mg / mL or higher. This is because the small amount of the first nuclease inhibited side reactions and reduced noise. Furthermore, the addition of the second nuclease prevented an increase in detection time. Furthermore, the addition of a second nuclease with low activity against the first invasive structure maintained the detection reaction and increased the signal-to-noise ratio (the difference in fluorescence intensity between a target RNA concentration of 30 pM or 300 pM and a target RNA concentration of 0 pM). These results, along with those of Example 2, indicate that a good signal-to-noise ratio can be achieved regardless of the concentration of the second nuclease, as long as the concentration ratio of the first nuclease to the second nuclease is within a certain range. In particular, favorable signal-to-noise ratios were obtained when the second nuclease concentrations were 0.008 mg / mL, 0.015 mg / mL, 0.03 mg / mL, 0.06 mg / mL, and 0.12 mg / mL, and even more favorable signal-to-noise ratios were obtained when the second nuclease concentrations were 0.03 mg / mL and 0.06 mg / mL. However, even if the concentration ratio of the second nucleic acid cleaving enzyme to the first nucleic acid cleaving enzyme is the same, if the concentrations of the first nucleic acid cleaving enzyme and the second nucleic acid cleaving enzyme are increased too much, the signal / noise ratio is not good, and when the concentration of the second nucleic acid cleaving enzyme is 0.24 mg / mL, a sufficient signal / noise ratio is not obtained.

[0118] Example 5 In this example, the effect of improving the signal-to-noise ratio in confirming the fluorescent signal from well 3 was confirmed.

[0119] (conditions) A reaction solution was prepared in a total volume of 20 μL per reaction, consisting of target RNA (0 pM, 30 pM), first nucleic acid (1 μM), second nucleic acid (1 μM), fourth nucleic acid (2 μM), trishydroxymethylaminomethane-HCl buffer (pH 9.0, 10 mM), MgCl2 (10 mM), Tween 20 (0.05% v / v), varying concentrations of 5'-nuclease (first nuclease cleavage enzyme) (0.045, 0.09, 0.18, 0.27, 0.36, 0.45 mg / mL), Flapendo Nuclease 1 (second nuclease cleavage enzyme) (0.03 mg / mL), and distilled water.

[0120] (procedure) The reaction solution was pumped into the first through-hole 15A of the microfluidic device 1, and then 80 μl of FC-40 (Sigma), which is immiscible with the detection reaction reagent and acts as a sealing liquid, was pumped through the first through-hole 15A to divide and seal the reagent into each well 3. This was heated on a hot plate at 66°C for 15 minutes to carry out the invader reaction. Next, a fluorescence microscope (manufactured by Keyence) was used to detect the fluorescence from each micropore using a NIBA fluorescence filter.

[0121] (result) The results are shown in Figure 10. The exposure time was 1000 msec. Figure 10A shows the result when the target RNA concentration was 30 pM, and a fluorescent signal with an intensity different from the noise in well 3 was confirmed. Figure 10B shows the result when the target RNA concentration was 0 pM, and it can be seen that the fluorescent intensity of the noise in well 3 was low.

[0122] Comparative Example 3 To confirm the extent of the side reaction, the behavior of a reaction solution in which the fourth nucleic acid, the first nucleic acid, and the first nucleic acid cleaving enzyme 5'-nuclease were mixed was observed in the absence of target RNA.

[0123] (conditions) A reaction solution was prepared in a total volume of 10 μL per reaction, consisting of the first nucleic acid (1 μM), the fourth nucleic acid (2 μM), 3-morpholinopropanesulfonic acid (pH 7.9, 10 mM), MgCl (10 mM), Tween 20 (0.05% v / v), 5'-nuclease (0.09 mg / mL) as the first nucleic acid cleaving enzyme, and distilled water.

[0124] (procedure) The reaction solution was placed in a micro test tube and heated at 65°C for 60 minutes in a real-time PCR device, and the change in fluorescence intensity (excitation: 490 nm, emission: 520 nm) was measured.

[0125] (result) The results are shown in Figure 11. It was observed that the fluorescence intensity increased over time even in the absence of target RNA. This result confirmed that at least the first nucleic acid and the fourth nucleic acid contributed to the increase in noise (increased fluorescence intensity even in the absence of target RNA).

[0126] Comparative Example 4 To confirm the extent of the side reaction, the behavior of a reaction solution in which the fourth nucleic acid, the first nucleic acid, and the second nucleic acid cleaving enzyme FEN-1 were mixed was observed in the absence of target RNA.

[0127] (conditions) A reaction solution was prepared in a total volume of 10 μL per reaction, consisting of the first nucleic acid (1 μM), the fourth nucleic acid (2 μM), 3-morpholinopropanesulfonic acid (pH 7.9, 10 mM), MgCl (10 mM), Tween 20 (0.05% v / v), Flapendo Nuclease 1 (0.03 mg / mL) as a second nucleic acid cleaving enzyme, and distilled water.

[0128] (procedure) The reaction solution was placed in a micro test tube and heated at 65°C for 60 minutes in a real-time PCR device, and the change in fluorescence intensity (excitation: 490 nm, emission: 520 nm) was measured.

[0129] (result) The results are shown in Figure 12. It was observed that the fluorescence intensity increased over time even in the absence of target RNA. However, it was confirmed that the contribution of noise was small compared to Figure 11. This result also confirmed that at least the first nucleic acid and the fourth nucleic acid contributed to the increase in noise (increased fluorescence intensity even in the absence of target RNA). The factors that affect the increase in noise are the first nucleic acid, the fourth nucleic acid, and the enzyme, with the first nucleic acid cleaving enzyme having a greater effect than the second nucleic acid cleaving enzyme. While it is easy to imagine increasing the amount of the first nucleic acid cleaving enzyme in an attempt to shorten the time required for detecting the target nucleic acid, conversely, decreasing the amount of the first nucleic acid cleaving enzyme can increase the signal-to-noise ratio during detection, enabling more effective detection of the target nucleic acid. On the other hand, adding the second nucleic acid cleaving enzyme can also prevent the detection time from increasing.

[0130] Although the preferred embodiment of the present invention has been described above, the present invention is not limited to the above embodiment. Addition, omission, substitution, and other modifications of the configuration are possible within the scope of the spirit of the present invention. [Industrial Applicability]

[0131] According to the present invention, it is possible to provide a nucleic acid detection method for quickly detecting a target nucleic acid from a fluid containing the target nucleic acid, and to provide a detection reagent for carrying out the nucleic acid detection method. [Explanation of symbols]

[0132] 1. Microfluidic Device 3 wells (microspaces) 4. Microwell array 5 Cover member 6 Opening surface 7 Peripheral Areas 8. Well formation area 9 Bottom 15 through holes 15A First through hole 15B Second through hole 16 Small hole 17 Large hole 20 fluid 23 Detection reagent (microdroplets)

Claims

1. A detection kit for detecting a target nucleic acid from a fluid containing the target nucleic acid, comprising: a first nucleic acid cleaving enzyme and a second nucleic acid cleaving enzyme; the target nucleic acid is RNA; the first nuclease is a flap endonuclease modified to recognize an invasion structure in an RNA / DNA double-stranded structure, and the second nuclease is FEN-1; further comprising a first nucleic acid and a second nucleic acid having a first flap portion; at least the first flap portion of the first nucleic acid and the second nucleic acid comprise DNA; the first nucleic acid and the second nucleic acid hybridize to a target nucleic acid to form a complex, thereby forming a first invasion structure, and a first flap site associated with the first invasion structure is cleaved by a first nucleic acid cleaving enzyme to generate a third nucleic acid; a fourth nucleic acid having a second flap site and a base sequence to which the third nucleic acid can hybridize; the fourth nucleic acid comprises DNA; a second invasion structure is formed by the third nucleic acid and the fourth nucleic acid forming a complex, and a second flap site associated with the second invasion structure is cleaved by a second nucleic acid cleaving enzyme to generate a cleavage target; the cleavage activity of the second nucleic acid cleaving enzyme on the first invasion structure is smaller than the cleavage activity of the first nucleic acid cleaving enzyme on the first invasion structure; A detection kit, wherein the cleavage activity of the first nucleic acid cleaving enzyme on the second invasion structure is lower than the cleavage activity of the second nucleic acid cleaving enzyme on the second invasion structure.

2. further comprising a fifth nucleic acid; the fifth nucleic acid comprises DNA; The detection kit of claim 1, wherein the third nucleic acid, the fourth nucleic acid, and the fifth nucleic acid form a complex to form a second invasion structure, and the second flap site associated with the second invasion structure is cleaved by a second nucleic acid cleaving enzyme to produce a cleavage target.

3. The detection kit according to claim 1 or 2, wherein at least one of the first nucleic acid and the fourth nucleic acid forming the invasion structure is fluorescently labeled.

4. The detection kit according to any one of claims 1 to 3, further comprising a microwell array.

5. The detection kit according to claim 4, wherein the height of the wells of the microwell array is 10 nm to 100 μm.

6. The density of the wells of the microwell array is 100,000 to 10,000,000 / cm ２ The detection kit according to claim 4 or 5,

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