Method for determining the presence or absence of very low concentrations of oligonucleotides, and method for determining whether a test subject has a specific disease or is at risk for a specific disease

Abstract

To provide a method for determining the presence or absence of a specific oligonucleotide having an extremely low concentration (1.0 pM or less) in a test sample.SOLUTION: (1) Provided is a method comprising preparing a test sample that may contain a probe having a sequence complementary to one or more specific oligonucleotides as well as at least one oligonucleotide among the specific oligonucleotides at a concentration of 1.0 aM to 1.0 pM so that the ratio of the concentration of the probe to the concentration of the at least one oligonucleotide in the test sample is 1.0×104:1 to 1.0×109:1 when the test sample contains 1.0aM to 1.0pM of the at least one oligonucleotide, followed by obtaining current aging data using a nanopore.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to a method for determining the presence or absence of an oligonucleotide at an extremely low concentration, and a method for determining whether a test subject has a specific disease or has a risk of developing a specific disease.

Background Art

[0002] Liquid biopsy is known as a method for detecting a specific oligonucleotide or the like of interest from a body fluid sample such as blood. Liquid biopsy has been attracting attention as a method for detecting a plurality of types of oligonucleotides such as microRNA that are differentially expressed when suffering from cancer, for example.

[0003] Nanopore analysis technology is known as a technology for detecting a specific oligonucleotide. Here, the nanopore analysis technology is a technology for analyzing a biomolecule based on a change in current observed when the biomolecule passes through a nanopore-sized through-hole in a protein.

[0004] For example, Patent Document 1 and Patent Document 2 disclose a method for detecting an oligonucleotide based on nanopore analysis technology using a probe that contains a sequence complementary to a target oligonucleotide and has a terminal extension at the 3'-end, 5'-end, or both ends thereof.

[0005] For example, Patent Document 3 discloses a method for simply and accurately determining the presence or absence of two or more specific oligonucleotides.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

SUMMARY OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION

[0007] However, none of Patent Documents 1 to 3 described above disclose a method for detecting a specific oligonucleotide at an extremely low concentration (1.0 pM or less) in a test sample. Therefore, conventionally, it has been difficult to perform a clinical test using the specific oligonucleotide present at an extremely low concentration as an indicator.

[0008] In addition to the method using the nanopore described above, methods using RT-qPCR or microarray are known as methods for detecting specific oligonucleotides. However, in any of these methods, when the specific oligonucleotide to be detected is at an extremely low concentration, the nucleic acid amplification efficiency is significantly low, and it is difficult to design primers for nucleic acid amplification of a specific oligonucleotide having a short base length, resulting in a problem of cross-hybridization.

[0009] However, if an oligonucleotide present at an extremely low concentration can be detected, it becomes possible to detect the presence of a disease in which the marker oligonucleotide is expressed only in trace amounts, and it is also considered possible to detect the presence of a progressive disease at an earlier stage.

[0010] The present disclosure has been made in view of the above, and an object thereof is to provide a method for determining the presence or absence of a specific oligonucleotide at an extremely low concentration (1.0 pM or less) in a test sample.

MEANS FOR SOLVING THE PROBLEMS

[0011] Specific means for solving the problems include the following aspects. <1> (1) A probe having a sequence complementary to one or more specific oligonucleotides, and a test sample that may contain at least one of the specific oligonucleotides at a concentration of 1.0 aM to 1.0 pM. When the test sample contains the at least one oligonucleotide at 1.0 aM to 1.0 pM, the ratio of the concentration of the probe to the concentration of the at least one oligonucleotide in the test sample is 1.0×10 4 :1 to 1.0×10 9 :1, and a first step of preparing the test sample so that the ratio is 1; (2) Applying a voltage between the test sample as the first solution and a second solution separated from each other by a lipid bilayer having a nanopore, and measuring the current intensity of the current flowing between the first solution and the second solution over time to obtain current change data over time; (3) A third step of determining the presence or absence of the at least one oligonucleotide in the test sample based on the current change data over time; A method for determining the presence or absence of at least one oligonucleotide at a concentration of 1.0 aM to 1.0 pM in a test sample, comprising the steps described above. <2> The method according to <1>, further comprising determining the amount of the at least one oligonucleotide. <3> The method according to <1> or <2>, wherein the one or more specific oligonucleotides include two or more specific oligonucleotides. <4> The method according to <3>, wherein the probe contains, in one molecule, all sequences complementary to each of the two or more specific oligonucleotides. <5> The order of the sequences complementary to the two or more specific oligonucleotides in the probe is selected so that the free energy of the complex of the probe and the two or more specific oligonucleotides is minimized. The method according to <3> or <4>. <6> The method according to <3>, wherein the probe contains two or more probes complementary to different specific oligonucleotides. <7> Between the first step and the second step, including a step of annealing the test sample, the method according to any one of <1> to <6> above. <8> The specific oligonucleotide is miRNA, and the probe is an oligodeoxyribonucleotide, the method according to any one of <1> to <7> above. <9> The probe has a (n) x structure at the 3'-end, where n is an arbitrary deoxyribonucleotide, x is an integer from 3 to 30, and x n's are identical to each other, the method according to any one of <1> to <8> above. <10> The (n) x structure is a polydeoxycytosine structure, the method according to <9> above. <11> The probe has a hairpin structure at the 5'-end, the method according to any one of <1> to <10> above. <12> (1-1) A probe having a sequence complementary to one or more specific oligonucleotides differentially expressed in a diseased person having a specific disease or an individual or tissue at risk of developing a specific disease, and a sample derived from a test subject containing at least one of the specific oligonucleotides, are mixed so that the concentration of the at least one oligonucleotide in the test sample is 1.0 aM to 1.0 pM and the ratio of the concentration of the probe to the concentration of the at least one oligonucleotide is 1.0×10 4 :1 to 1.0×10 9 :1 to obtain a test sample, or, (1-2) A probe having a sequence complementary to one or more specific oligonucleotides that are differentially expressed in a subject having a specific disease, an individual having a risk of developing a specific disease, or their tissue, and a sample derived from a test subject containing at least one of the specific oligonucleotides are mixed in a test sample such that the concentration of the at least one oligonucleotide in the test sample is 1.0 aM to 1.0 pM and the ratio of the concentration of the probe to the concentration of the at least one oligonucleotide is 1.0×10 4 :1 to 1.0×10 9 :1 to obtain a test sample. A first step, (2) Applying a voltage between the test sample as the first solution and a second solution separated from each other by a lipid bilayer having a nanopore, and measuring the current intensity of the current flowing between the first solution and the second solution over time to obtain current change data over time. (3) Detecting the presence of the at least one oligonucleotide in the test sample based on the current change data over time, and thereby determining whether the test subject has a specific disease or has a risk of developing a specific disease. A method for determining whether a test subject has a specific disease or has a risk of developing a specific disease, comprising: <13> The method according to <12>, wherein the one or more specific oligonucleotides comprise two or more specific oligonucleotides. <14> The method according to <12> or <13>, wherein the specific disease is cholangiocarcinoma.

Advantages of the Invention

[0012] According to the present disclosure, a method for determining the presence or absence of a specific oligonucleotide at an extremely low concentration in a test sample can be provided.

Brief Description of the Drawings

[0013]

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Mode for Carrying Out the Invention

[0014] Hereinafter, embodiments for carrying out the present disclosure will be described in detail. However, the present disclosure is not limited to the following embodiments. The following embodiments are exemplary, and their components (including element steps, etc.) are not essential unless otherwise explicitly stated. The same applies to numerical values and their ranges, which do not limit the present disclosure. Also, within the scope of the technical idea of the present disclosure, various changes and modifications can be made by those skilled in the art. In the numerical range indicated by "~" in the present disclosure, the numerical values described before and after "~" are included as the minimum value and the maximum value, respectively. In the numerical ranges described stepwise in the present disclosure, the upper limit value or the lower limit value described in one numerical range may be replaced with the upper limit value or the lower limit value of another numerically described stepwise range. Also, in the numerical ranges described in the present disclosure, the upper limit value or the lower limit value of the numerical range may be replaced with the value shown in the examples. In the present disclosure, the term "step" includes not only steps independent of other steps but also steps that, even if not clearly distinguishable from other steps, are included if the purpose of the step is achieved. In the present disclosure, each component may contain a plurality of corresponding substances. When there are a plurality of substances corresponding to each component, the content rate of each component means the total content rate of the plurality of substances, unless otherwise specified. Also, it is obvious to those skilled in the art that the present disclosure can be implemented without using some or all of the specific and detailed content described in the present disclosure. Also, in order to avoid obscuring aspects of the present disclosure, detailed explanations or illustrations of well-known points may be omitted in some cases. In addition, the sizes of the members in the drawings are conceptual, and the relative relationships of the sizes between the members are not limited thereto. Also, members having substantially the same function may be given the same reference numerals throughout the drawings, and duplicate explanations may be omitted.

[0015] ≪Method for Determining the Presence or Absence of at Least One Oligonucleotide at a Concentration of 1.0 aM to 1.0 pM in a Test Sample (First Determination Method)≫ The method for determining the presence or absence of an oligonucleotide at an extremely low concentration according to the present disclosure (hereinafter, also referred to as the first determination method according to the present disclosure) is as follows. (1) A first step of preparing a probe having a sequence complementary to one or more specific oligonucleotides and a test sample that may contain at least one of the specific oligonucleotides at a concentration of 1.0 aM to 1.0 pM such that when the test sample contains the at least one oligonucleotide at 1.0 aM to 1.0 pM, the ratio of the concentration of the probe to the concentration of the at least one oligonucleotide in the test sample is 1.0×10 4 :1 to 1.0×10 9 :1; (2) A second step of applying a voltage between the test sample as the first solution and a second solution separated from each other by a lipid bilayer having a nanopore, and measuring the current intensity of the current flowing between the first solution and the second solution over time to obtain current change data over time; (3) A third step of determining the presence or absence of the at least one oligonucleotide in the test sample based on the current change data over time. It has the above.

[0016] As described above, by the method having the first to third steps described in (1) to (3) above, the presence or absence of at least one oligonucleotide at a concentration of 1.0 aM to 1.0 pM in a test sample can be determined.

[0017] Although the mechanism of the first determination method according to the present disclosure is not clear, it is presumed as follows.

[0018] The flow of the nanopore analysis technology according to the present disclosure will be described with reference to the drawings. FIG. 1 shows a schematic cross-sectional view of an example of an analyzer used in the nanopore analysis technology according to the present disclosure. As shown in FIG. 1, this analyzer includes, for example, a nanopore 1N, a lipid bilayer 2, a first solution 3, a second solution 4, a specific oligonucleotide 5A (or specific oligonucleotides 5A and 5B) contained in the first solution (test sample), a probe 6, and a voltage application means 7. Further, electrolytes are contained in the first solution 3 and the second solution 4. The first solution and the second solution are housed in a container. Examples of the analyzer used in the nanopore analysis technology include the analyzers disclosed in JP-A-2015-77559 and JP-A-2014-100672.

[0019] The first solution 3 and the second solution 4 are separated from each other by the lipid bilayer 2. The lipid bilayer 2 has a nanopore 1N that penetrates perpendicularly to the thickness direction of the lipid bilayer 2. The nanopore 1N has a through-hole 1P that penetrates perpendicularly to the thickness direction of the lipid bilayer 2.

[0020] A solution in which the probe 6 and the specific oligonucleotide 5A (or specific oligonucleotides 5A and 5B) are mixed constitutes the first solution 3. In the first solution (test sample), the probe 6 hybridizes with the specific oligonucleotide 5A in a region in the probe 6 to form a complex HYB1. Alternatively, the probe 6 hybridizes with two different specific oligonucleotides 5A and 5B in different regions in the probe 6 to form a complex HYB2. Note that FIG. 1 is a schematic cross-sectional view when the complex HYB2 is formed. The following description of the flow of the nanopore analysis technology according to the present disclosure is an explanation when the complex HYB2 is formed.

[0021] The voltage application means 7 is connected to each of the first solution 3 and the second solution 4 so as to be able to apply a voltage between the first solution 3 and the second solution 4 (that is, via the lipid bilayer membrane 2). Although not shown, electrodes are provided at the tips of the voltage application means 7 connected to each of the first solution 3 and the second solution 4. Further, the voltage application means 7 may be electrically connected to a power source. The voltage application means 7 may be electrically connected to a voltmeter.

[0022] The complex HYB2 enters the through-hole 1P of the nanopore 1N from the first solution 3 and proceeds in the direction of the second solution 4 by the application of a voltage. At this time, the pore size of the through-hole 1P is such that only single-stranded oligonucleotides can pass through. Therefore, as the complex HYB2 moves in the through-hole 1P in the direction of the second solution 4 by the application of a voltage, it dissociates from the double-stranded form with the specific oligonucleotides 5A and 5B and becomes a single-stranded form of the probe 6 while proceeding in the through-hole 1P in the direction of the second solution. Finally, the probe 6 passes through the through-hole 1P and moves into the second solution 4.

[0023] Next, the change in current accompanying this nanopore analysis will be described. For example, when a voltage is applied between the first solution 3 and the second solution 4 by the voltage application means 7, a current passing through the through-hole 1P is observed. Next, when the complex HYB2 enters the through-hole 1P, the passage of current in the through-hole 1P is inhibited, and a decrease in current intensity is observed. Then, when the complex HYB2 that has entered the through-hole 1P dissociates from the complex with the specific oligonucleotides 5A and 5B and the probe 6 passes through the through-hole 1P, the current that had been inhibited from passing through the through-hole 1P flows again, and the current intensity increases again. Note that the phenomenon of current intensity fluctuation when passing through the through-hole 1P is also observed when only the probe 6 passes through.

[0024] FIG. 2 shows a graph representing an example of current change data over time in the nanopore analysis technique according to the present disclosure. As shown in FIG. 2, in the current change data over time, the time T during which the current passing through the through-hole 1P of the nanopore 1N is not inhibited Oand the time T during which the current passing through the through-hole 1P of the nanopore 1N is inhibited C is observed. In the nanopore analysis technique of the present disclosure, the time T during which the current passing through the through-hole 1P of the nanopore 1N is inhibited C (hereinafter also referred to as "Unzipping time" or "interval time") is read from the above-described current change data over time, and based on this data, the presence or absence of a specific oligonucleotide in the test sample is determined.

[0025] FIGS. 3(A-1) to (C-1) show conceptual diagrams illustrating an example of the interaction between each complex and the nanopore. Note that the description of the reference numerals in FIG. 3 can be applied to the description of the same reference numerals in FIG. 1. FIG. 3(C-1) is an example of the interaction between the nanopore 1N and the complex HYB5 when all five specific oligonucleotides 5A to 5E hybridize to the probe 6 having a sequence complementary to the five specific oligonucleotides 5A to 5E to form the complex HYB5. As shown in FIG. 3(C-1), upon application of a voltage, the HYB5 that has entered the through-hole 1P of the nanopore 1N is considered to move in the direction of the second solution 4 and gradually dissociate from the specific oligonucleotides 5A to 5E.

[0026] FIG. 3(B-1) is an example of the interaction between the nanopore 1N and the complex HYB3 when three specific oligonucleotides 5A, 5C, and 5E hybridize to the probe 6 having a sequence complementary to the five specific oligonucleotides 5A to 5E to form the complex HYB3. As shown in FIG. 3(B-1), similar to HYB5, upon application of a voltage, the HYB3 that has entered the through-hole 1P of the nanopore 1N is considered to move in the direction of the second solution 4 and gradually dissociate from the specific oligonucleotides 5A to 5E.

[0027] Figure 3(A-1) shows an example of the interaction between nanopore 1N and complex HYB1 when only one specific oligonucleotide 5E out of five specific oligonucleotides 5A to 5E hybridizes to probe 6 having a sequence complementary thereto to form complex HYB1. As shown in Figure 3(A-1), similar to HYB5, upon application of a voltage, HYB1 that has entered the through-hole 1P of nanopore 1N is considered to move in the direction of the second solution 4 and to dissociate from the complex with the specific oligonucleotide 5E.

[0028] When the respective behaviors of the above complexes HYB5, HYB3, and HYB1 in the nanopore are captured as current change data over time, as shown in Figures 3(A-2) to (C-2), the interval time INT-HYB5 of HYB5 is longer than the interval time INT-HYB3 of HYB3 and the interval time INT-HYB1 of HYB1. Also, the interval time INT-HYB3 of HYB3 is longer than the interval time INT-HYB1 of HYB1 and shorter than INT-HYB5. That is, the interval time is affected by the combination of complexes formed by probe 6. Also, the interval time becomes longer as the number of specific oligonucleotides that hybridize to probe 6 increases. Here, the specific oligonucleotide refers to the oligonucleotide to be measured that hybridizes to probe 6, and its sequence is not particularly limited.

[0029] However, in practice, it is difficult to determine the presence or absence of a specific oligonucleotide at an extremely low concentration (1.0 pM or less) in a test sample. The factors include that simply including the probe in the test sample results in a very low probability that the specific oligonucleotide contained in the test sample forms a complementary strand with the probe when the specific oligonucleotide is at an extremely low concentration, and further, the probability (passage frequency) that the complementary strand passes through the nanopore is also very low. For the above reasons, it has been common technical knowledge that in conventional oligonucleotide detection using a nanopore, it is only possible to determine the presence or absence of an oligonucleotide at a concentration higher than 1.0 pM.

[0030] On the other hand, in the first determination method according to the present disclosure, the presence or absence of a specific oligonucleotide at an extremely low concentration (1.0 pM or less) in a test sample can be determined. Specifically, in the first determination method according to the present disclosure, a probe having a sequence complementary to one or more specific oligonucleotides, and a test sample that may contain at least one of the specific oligonucleotides at a concentration of 1.0 aM to 1.0 pM are used. When the test sample contains at least one of the oligonucleotides at 1.0 aM to 1.0 pM, the ratio of the concentration of the probe to the concentration of the at least one oligonucleotide in the test sample is 1.0×10 4 :1 to 1.0×10 9 :1. By preparing in this way, surprisingly, when at least one oligonucleotide at 1.0 aM to 1.0 pM is contained in the test sample, the detection of the oligonucleotide is possible. If the ratio of the concentration of the probe to the concentration of the at least one oligonucleotide in the test sample exceeds 1:1, the probe is already in excess. The fact that the above effect is particularly obtained in the case of a concentration ratio of 1.0×10 4 :1 to 1.0×10 9 :1 could not be predicted from the common general knowledge of the prior art. Further, even if the concentration ratio is made such that the probe is overwhelmingly in excess as described above, the fact that the oligonucleotide only exists at a low concentration of 1.0 aM to 1.0 pM remains unchanged. From this, too, the fact that the above effect can be obtained could not be predicted from the common general knowledge of the prior art. The above effect is presumably because when the concentration ratio is 1.0×10 4 :1 to 1.0×10 9 :1, the probability that the probe and the specific oligonucleotide form a complementary strand becomes particularly high. Note that the present disclosure is not limited to the above estimation mechanism at all.

[0031] The first determination method of the present disclosure is (1) A probe having a sequence complementary to one or more specific oligonucleotides, and a test sample that may contain at least one of the specific oligonucleotides at a concentration of 1.0 aM to 1.0 pM. When the test sample contains the at least one oligonucleotide at 1.0 aM to 1.0 pM, the ratio of the concentration of the probe to the concentration of the at least one oligonucleotide in the test sample is 1.0×10 4 :1 to 1.0×10 9 :1, and a first step of preparing them; (2) A voltage is applied between the test sample as the first solution and the second solution, which are separated from each other by a lipid bilayer having a nanopore, and the current intensity of the current flowing between the first solution and the second solution is measured over time to obtain current change data over time. A second step; (3) A third step of determining the presence or absence of the at least one oligonucleotide in the test sample based on the current change data over time; and includes.

[0032] The first determination method of the present disclosure may further include determining the amount of the at least one oligonucleotide.

[0033] Hereinafter, each step will be described in detail.

[0034] (First step) The first determination method according to the present disclosure includes a first step. In the first step, a probe having a sequence complementary to one or more specific oligonucleotides, and a test sample that may contain at least one of the specific oligonucleotides at a concentration of 1.0 aM to 1.0 pM. When the test sample contains the at least one oligonucleotide at 1.0 aM to 1.0 pM, the ratio of the concentration of the probe to the concentration of the at least one oligonucleotide in the test sample is 1.0×10 4 :1 to 1.0×10 9 :1, and prepare them.

[0035] - Test sample - The test sample according to the present disclosure may or may not contain a specific oligonucleotide. When the first determination method according to the present disclosure is used for the test sample, the presence of the specific oligonucleotide can be detected when the test sample contains the specific oligonucleotide at an extremely low concentration (1.0 pM or less).

[0036] The test sample is a sample that may contain at least one of the specific oligonucleotides at a concentration of 1.0 aM to 1.0 pM, but the lower limit value of the concentration may be 10 aM, 100 aM, 0.5 fM, 1.0 fM, 5.0 fM, 10 fM, 20 fM, or 50 fM. The upper limit value of the concentration is not limited to 1.0 pM, and may be, for example, 800 fM, 500 fM, 300 fM, 200 fM, 100 fM, 50 fM, 20 fM, or 10 fM. For example, the test sample may be a sample that may contain at least one of the specific oligonucleotides at a concentration of 10 aM to 100 fM. Note that these upper and lower limit values can be freely combined with any of the above as long as there is no contradiction.

[0037] In the first determination method according to the present disclosure, the test sample that may contain at least one of the specific oligonucleotides at a concentration of 1.0 aM to 1.0 pM only needs to be a test sample that may contain at least one of the specific oligonucleotides at a concentration of 1.0 aM to 1.0 pM, and the inclusion / non-inclusion and concentration of the remaining oligonucleotides among the specific oligonucleotides are not particularly limited. Also, the test sample only needs to possibly contain at least one of the specific oligonucleotides at a concentration of 1.0 aM to 1.0 pM at the time of measurement, and it may be found later that the test sample does not contain the at least one specific oligonucleotide, contains it at a concentration less than 1.0 aM, or contains it at a concentration exceeding 1.0 pM.

[0038] In the first determination method according to the present disclosure, when the test sample contains the at least one kind of oligonucleotide at 1.0 aM to 1.0 pM, the ratio of the concentration of the probe to the concentration of the at least one kind of oligonucleotide in the test sample is 1.0×10 4 :1 to 1.0×10 9 :1. It is not required to confirm in advance before measurement that it is such a concentration, and it is sufficient if it can be confirmed afterwards that the above concentration ratio is satisfied. The post hoc confirmation of the concentration of the oligonucleotide can be obtained by, if necessary, one embodiment of the first determination method according to the present disclosure, or can also be obtained by other methods such as real-time PCR. The first determination method according to the present disclosure determines the presence or absence of an oligonucleotide at an extremely low concentration. If it can be confirmed that the above concentration ratio is satisfied, even if it is a post hoc confirmation, it can be understood that the presence or absence of an oligonucleotide at an extremely low concentration has been determined by the first determination method according to the present disclosure.

[0039] The sample from which the test sample is derived may be solid or liquid. When the sample is solid, for example, it is preferable to use a dilution obtained by suspending, dispersing or dissolving the solid in a solution medium. The solution medium is not particularly limited, and examples thereof include water or a buffer solution.

[0040] Examples of the sample include samples derived from a subject to be examined such as a human or a non-human animal (mammals other than humans). Examples of the sample derived from the subject to be examined include biological specimens. The biological specimen is not particularly limited, and examples thereof include urine, blood, or saliva. Examples of the blood specimen include red blood cells, whole blood, serum, or plasma. The sample obtained from the inspection target may be liquid or solid. For example, the undiluted liquid of the sample obtained from the inspection target may be used as it is, or a diluted liquid obtained by suspending, dispersing or dissolving the sample in a medium may be used. When the sample obtained from the inspection target is solid, for example, it is preferable to use a diluted liquid obtained by suspending, dispersing or dissolving the specimen in a solution medium as the liquid specimen. The solution medium is not particularly limited, and examples thereof include water or a buffer solution. For example, as the test sample as the first solution, when the sample is liquid, the sample may be used as the test sample as it is, or a sample diluted in a pH buffer solution or the like may be used as the test sample. The liquid specimen thus obtained can be used as the first solution or added to the first solution and used in the first determination method according to the present disclosure.

[0041] -Specific oligonucleotide- The specific oligonucleotide according to the present disclosure represents an oligonucleotide having a specific sequence to be detected.

[0042] The specific oligonucleotide is preferably a single-stranded oligonucleotide in that it easily hybridizes with a probe to form a complex. For example, a single-stranded oligonucleotide that is differentially expressed or not expressed depending on the presence or absence of a specific disease, that is, a single-stranded oligonucleotide whose expression is differential with respect to the presence or absence of a specific disease, may be selected as the specific oligonucleotide.

[0043] The specific oligonucleotide is preferably a single-stranded oligonucleotide having a base length of 15 or more and 30 or less. The single-stranded oligonucleotide having a base length of 15 or more and 30 or less may be, for example, single-stranded DNA or single-stranded RNA such as microRNA (miRNA). Among the above, as the specific oligonucleotide, for example, from the viewpoint of determining the presence or risk of suffering from a specific disease, it is preferably miRNA. When the specific oligonucleotide is miRNA, the probe can be an oligodeoxyribonucleotide having a sequence complementary to the miRNA.

[0044] One or more specific oligonucleotides according to the present disclosure may be two or more specific oligonucleotides, three or more specific oligonucleotides, or five or more specific oligonucleotides from the viewpoint of being able to determine the presence or absence of a plurality of oligonucleotides at once, or from the viewpoint of being able to more precisely determine the presence or risk of a specific disease.

[0045] In addition, when there are multiple types of specific oligonucleotides according to the present disclosure, if there is a possibility that at least one of the specific oligonucleotides is contained in a test sample at a concentration of 1.0 aM to 1.0 pM, the first determination method according to the present disclosure can be applied. For example, when there are five specific oligonucleotides according to the present disclosure, if there is a possibility that, for example, two of the specific oligonucleotides are contained in a test sample at a concentration of 1.0 aM to 1.0 pM, the other three specific oligonucleotides may be contained at a concentration higher than 1.0 pM. Even in such a case, the first determination method according to the present disclosure can be applied.

[0046] -Probe- The probe according to the present disclosure is not particularly limited as long as it is a single-stranded polynucleotide having a sequence complementary to one or more specific oligonucleotides. The probe may contain a sequence other than the sequence complementary to the specific oligonucleotide.

[0047] The "complementary sequence" refers to a sequence having a complementarity of a predetermined degree or more with respect to the entire length of the sequence of each specific oligonucleotide in the test sample.

[0048] The complementarity of each of the one or more specific oligonucleotides in the probe is not particularly limited as long as the probe can hybridize with at least one of the specific oligonucleotides to obtain a complex. For example, the probe may contain a sequence having 80% or more complementarity to the sequence of each specific oligonucleotide, may contain a sequence having 90% or more complementarity, may contain a sequence having 95% or more complementarity, or may contain a sequence having 100% or more complementarity (i.e., a sequence completely complementary to the full length of the specific oligonucleotide). Alternatively, the probe may contain a sequence obtained by adding, deleting, or substituting 5 bases or less with respect to the sequence completely complementary to each specific oligonucleotide. The number of added, deleted, or substituted bases of the probe with respect to the sequence completely complementary to each specific oligonucleotide is preferably 3 or less, more preferably 1 or less, and even more preferably 0.

[0049] From the viewpoint of reducing non-specific hybridization and increasing the efficiency of specific hybridization, the probe preferably has a sequence completely complementary to each of the base sequences of the specific oligonucleotides.

[0050] By using a probe having a sequence completely complementary to each of the base sequences of the specific oligonucleotides, for example, for a plurality of complexes in which the number of specific oligonucleotides hybridized to the probe is different, a greater difference can be made for each interval time.

[0051] The probe having a sequence complementary to the one or more specific oligonucleotides may be a probe having a sequence complementary to two or more specific oligonucleotides, a probe having a sequence complementary to three or more specific oligonucleotides, or a probe having a sequence complementary to five or more specific oligonucleotides from the viewpoint of being able to determine the presence or absence of a plurality of oligonucleotides at once or from the viewpoint of being able to more precisely determine the presence or risk of a specific disease.

[0052] In the present disclosure, a probe having a sequence complementary to one or more specific oligonucleotides, and a test sample that may contain at least one of the specific oligonucleotides at a concentration of 1.0 aM to 1.0 pM are prepared such that when the test sample contains the at least one oligonucleotide at 1.0 aM to 1.0 pM, the ratio of the concentration of the probe to the concentration of the at least one oligonucleotide in the test sample is 1.0×10 4 :1 to 1.0×10 9 :1, but the lower limit value of the concentration ratio is 5.0×10 4 、1.0×10 5 、5.0×10 5 、1.0×10 6 、5.0×10 6 、1.0×10 7 、5.0×10 7 、or 1.0×10 8 may be, and the upper limit value of the concentration ratio is 5.0×10 8 、1.0×10 8 、5.0×10 7 、1.0×10 7 、5.0×10 6 、1.0×10 6 、5.0×10 5 、or 1.0×10 5 may be. Note that these upper limit and lower limit values can be freely combined with any of the above as long as there is no contradiction. Therefore, the concentration ratio may be, for example, 1.0×10 5 :1 to 1.0×10 8 :1, or 1.0×10 6 :1 to 1.0×10 7 :1 may be.

[0053] The concentration of the probe contained in the prepared test sample is not particularly limited as long as it satisfies the ratio of the concentration of the probe to the concentration of the at least one oligonucleotide in the test sample as described above. From the viewpoint of determining the presence or absence of a specific oligonucleotide at an extremely low concentration (1.0 pM or less) in the test sample, the concentration of the probe contained in the prepared test sample may be, for example, 10 fM to 1.0 mM. The lower limit value of the concentration of the probe may be 50 fM, 100 fM, 500 fM, 1.0 pM, 5.0 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1.0 nM, 5.0 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1.0 μM, 5.0 μM, 10 μM, 50 μM, or 100 μM. The upper limit value of the concentration of the probe may be 500 μM, 100 μM, 50 μM, 10 μM, 5.0 μM, 1.0 μM, 500 nM, 100 nM, 50 nM, 10 nM, 5.0 nM, 1.0 nM, 500 pM, 100 pM, 50 pM, 10 pM, 5.0 pM, 1.0 pM, or 500 fM. Note that these upper limit values and lower limit values can be freely combined with any of the above as long as there is no contradiction. For example, the concentration of the probe may be 1.0 pM to 100 μM, or 100 pM to 10 μM.

[0054] When the test sample contains the at least one oligonucleotide at 1.0 aM to 1.0 pM as used in the present disclosure refers to the conditions when the test sample contains the at least one oligonucleotide at 1.0 aM to 1.0 pM, and does not exclude the existence of other cases. Therefore, the test sample may or may not contain the at least one oligonucleotide at 1.0 aM to 1.0 pM.

[0055] The ratio of the concentration of the probe to the concentration of the at least one oligonucleotide in the test sample according to the present disclosure is 1.0×10 4 :1 to 1.0×10 9"Prepare so that it becomes 1:1" means that in the first step, the probe and the at least one kind of oligonucleotide in the test sample are mixed so that the concentration ratio is 1.0×10 4 :1 to 1.0×10 9 :1, or it may already be in a mixed state where the probe and the at least one kind of oligonucleotide in the test sample have a concentration ratio of 1.0×10 4 :1 to 1.0×10 9 :1 even before the first step.

[0056] The type of probe may be one kind or a plurality of kinds.

[0057] When the type of probe is one kind, the probe may be composed of a probe that contains all the sequences complementary to each of two or more specific oligonucleotides in one molecule. When the probe is a probe that contains all the sequences complementary to each of the two or more specific oligonucleotides in one molecule, it tends to be easier to adjust the interval time of various complexes formed by the two or more specific oligonucleotides and the probe.

[0058] The probe may be composed of two or more probes that are complementary to different specific oligonucleotides. When the type of probe is two or more kinds, it is not necessary for one probe to contain all of two or more sequences complementary to two or more specific oligonucleotides, and it is sufficient that the probe complementary to each specific oligonucleotide is included in any one or more of the two or more probes. For example, two or more probes having sequences complementary to specific oligonucleotides with different sequences may be used. In this case, the number of types of probes to be used can be the same as the number of types of target specific oligonucleotides. Also, when using two or more probes, the probe may be modified as necessary with polyethylene glycol or the like in order to adjust the difference in interval time between each probe.

[0059] The probe may be a plurality of probe groups each containing a sequence complementary to at least one of the two or more specific oligonucleotides. When the probe is a plurality of probe groups each containing a sequence complementary to at least one of the two or more specific oligonucleotides, it tends to be easier to adjust the interval time of various complexes formed between the two or more specific oligonucleotides and the probe by designing and / or modifying each probe.

[0060] In the probe, the sequence complementary to one specific oligonucleotide may be divided into a plurality of parts, and each part may be present in a separate probe. In this case, if the separate probes coexist, a complementary sequence is formed in a form spanning the probes. For example, Probe 3-1 and Probe 3-2 described later have a sequence complementary to miR-20a and a sequence complementary to miR-17-5a in a form connecting Probe 3-1 and Probe 3-2. Even in such a case, in the present disclosure, it is included in the scope of the "probe having a complementary sequence".

[0061] The order of the sequences complementary to the two or more specific oligonucleotides in the probe is preferably selected so that the free energy of the complex between the probe and the two or more specific oligonucleotides is minimized.

[0062] When the order of the sequences complementary to each specific oligonucleotide is selected so that the free energy of the complex between the probe and the two or more specific oligonucleotides is minimized, the complex formed by hybridization of the probe and the two or more specific oligonucleotides tends to exist stably. Therefore, the variation in the interval time in the nanopore analysis of each complex tends to be suppressed.

[0063] The free energy of the complex between the probe and the two or more specific oligonucleotides is determined by NUPACK of Caltech (California Institute of Technology).

[0064] In the present disclosure, the combination of the type of the specific oligonucleotide and the type of the probe is not particularly limited. For example, the specific oligonucleotide can be miRNA, and the probe can be oligodeoxyribonucleotide. When the specific oligonucleotide is miRNA and the probe is oligodeoxyribonucleotide, the presence or absence of a specific miRNA at an extremely low concentration (1.0 pM or less) in a test sample can be determined.

[0065] The probe may have a (n) x structure at the 3'-end, where n is an arbitrary deoxyribonucleotide, x is an integer from 3 to 30, and it is preferable that the x number of n are the same as each other. When the probe has a (n) x structure at the 3'-end, when a voltage is applied, the complex tends to easily enter the through-hole of the nanopore from the 3'-end side of the probe.

[0066] When the probe has a (n) x structure at the 3'-end, the (n) x structure is preferably a polydeoxycytosine structure. The polydeoxycytosine structure is not particularly limited as long as it is a polymer having deoxycytosine as a constituent unit. For example, for the purpose of preferably allowing the complex to exist in the through-hole of the nanopore, it is preferably polydeoxycytosine in which 20 deoxycytosines are linked, which is longer than the length in the thickness direction in the through-hole of the nanopore.

[0067] The probe may have a hairpin structure at the 5'-end. Since the hairpin structure is bulky, the 5'-end of the probe tends to be difficult to enter the through-hole of the nanopore. That is, when the probe has a hairpin structure at the 5'-end, the entry of the nanopore into the through-hole from the 5'-end side of the probe can be suppressed, and it can be made easier to enter the through-hole of the nanopore only from one direction on the 3'-end side. As a result, the variation in the interval time in the nanopore analysis of each complex tends to be suppressed.

[0068] (Annealing process) The first determination method according to the present disclosure may include a step of annealing the test sample between the first step and the second step described below.

[0069] When a step of annealing the test sample is performed between the first step and the second step described below, even if a mismatch occurs and an unstable complex is formed when the probe hybridizes with the specific oligonucleotide contained in the test sample in the test sample, it tends to be rearranged into a thermally more stable complex. Therefore, it tends to be possible to suppress unevenness in the dwell time of the obtained complex.

[0070] The annealing temperature is preferably 70°C or higher and 100°C or lower, more preferably 80°C or higher and 100°C or lower, and even more preferably 90°C or higher and 100°C or lower. The annealing time is preferably 1 minute or longer and 20 minutes or shorter, more preferably 1 minute or longer and 15 minutes or shorter, and even more preferably 1 minute or longer and 10 minutes or shorter.

[0071] (Second step) The first determination method according to the present disclosure includes a second step. In the second step, a voltage is applied between the test sample as the first solution and the second solution, which are separated from each other by a lipid bilayer having a nanopore, and the current intensity of the current flowing between the first solution and the second solution is measured over time to obtain current change data over time. Note that as the first solution, the test sample prepared in the first step or the test sample obtained by further annealing the test sample prepared in the first step can be used.

[0072] The nanopore is not particularly limited as long as it has a through-hole penetrating in the thickness direction of the lipid bilayer membrane, and an appropriate one such as an ion channel (for example, an α-hemolysin transmembrane protein such as α-hemolysin derived from Staphylococcus aureus), a synthetic product, etc. may be used. For example, from the viewpoint of having a pore diameter that suppresses the passage of double-stranded nucleic acids and allows only single-stranded nucleic acids to pass through, it is preferable to use an α-hemolysin transmembrane protein or an ion channel having a pore diameter comparable thereto.

[0073] The lipid forming the lipid bilayer membrane is not particularly limited as long as it can stably form a lipid bilayer membrane at room temperature, and an appropriate lipid may be used. Examples of the lipid forming the lipid bilayer membrane include phospholipids such as difthanoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), difthanoyl phosphatidylethanolamine (DPhPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Among the above, as the lipid forming the lipid bilayer membrane, from the viewpoint of obtaining a more stable lipid bilayer membrane at room temperature, difthanoyl-sn-glycero-3-phosphocholine (DPhPC) having a high phase transition temperature is preferable. Note that the lipid bilayer membrane may be configured to contain a steroid compound such as cholesterol as necessary.

[0074] As a method for producing the lipid bilayer membrane, known production methods such as those disclosed in JP-A-2015-77559 and JP-A-2014-10062 may be applied.

[0075] For nanopore analysis, a commercially available product may be used as long as it has the configuration of the present disclosure. Commercially available products capable of nanopore analysis include, for example, MinION, GridION X5 , SmidgION, PromethION, etc.

[0076] The ratio of the concentration of the probe having a sequence complementary to one or more specific oligonucleotides in the first solution to the concentration of at least one of the specific oligonucleotides is as described above. For example, it may be 1.0×10 4 :1 to 1.0×10 9 :1, or may be 1.0×10 5 :1 to 1.0×10 8 :1, or may be 1.0×10 6 :1 to 1.0×10 7 :1.

[0077] The concentration of the probe contained in the first solution is not particularly limited and may be appropriately set according to the scale of the first solution. Although the concentration of the probe is as described above, for example, from the viewpoint of adjusting the observation frequency of the interval time derived from each specific oligonucleotide, it may be 10 fM to 1.0 mM, may be 1.0 pM to 100 μM, or may be 100 pM to 10 μM.

[0078] The first solution and the second solution are not particularly limited as long as the current passing through the through-hole of the nanopore can be observed, and an appropriate electrolyte solution may be applied as appropriate. Examples of the type of electrolyte contained in the electrolyte solution include KCl and NaCl. When an electrolyte solution is used, the concentration of the electrolyte may be, for example, 1000 mM or more and 10 M or less from the viewpoint of obtaining a sufficient current intensity.

[0079] The types of the first solution and the second solution are not particularly limited as long as nanopore analysis is possible, and suitable solutions may be used as appropriate. For example, when the specific oligonucleotide is miRNA, as the first solution and the second solution, a pH buffer solution adjusted to a pH of 6.0 or more and 8.0 or less may be used. The pH buffer contained in the pH buffer solution is not particularly limited. For example, 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPSO), and 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS) etc. may be mentioned.

[0080] It is preferable that the first solution and the second solution are accommodated in a container, a well or the like.

[0081] The method of applying a voltage between the first solution and the second solution is not particularly limited as long as a voltage can be applied between the first solution and the second solution over time. For example, electrodes may be provided in each of the first solution and the second solution, and a voltage may be applied through these two electrodes. Further, the electrode may be electrically connected to a power source and a control device for controlling the voltage. The electrode may be electrically connected to a monitoring device for obtaining current change data over time.

[0082] The voltage applied between the first solution and the second solution is not particularly limited as long as the complex of the probe and the specific oligonucleotide can penetrate into the through-hole of the nanopore. For example, from the viewpoint of shortening the passing time of the through-hole while making it easy to determine each type of complex for the interval time of the complex of one or more specific oligonucleotides and the probe, the voltage applied between the first solution and the second solution is preferably 50 mV or more and 300 mV or less, and more preferably 100 mV or more and 200 mV or less.

[0083] (Third step) The first determination method according to the present disclosure includes a third step. In the third step, based on the current change over time data, the presence or absence of the at least one kind of oligonucleotide in the test sample is determined.

[0084] The third step according to the present disclosure is not particularly limited as long as it can determine the presence or absence of the at least one kind of oligonucleotide in the test sample based on the current change over time data. When the test sample contains the at least one kind of oligonucleotide, the current change over time data includes a signal specific to the binding of the at least one kind of oligonucleotide to the probe, and thus it can be determined that the at least one kind of oligonucleotide is present in the test sample. On the other hand, when the test sample does not contain the at least one kind of oligonucleotide, the current change over time data does not include data specific to the binding of the at least one kind of oligonucleotide to the probe, and thus it can be determined that the at least one kind of oligonucleotide is not present in the test sample. Furthermore, based on the frequency of the signal specific to the binding of the at least one kind of oligonucleotide to the probe in the current change over time data, the amount of the at least one kind of oligonucleotide in the test sample can also be determined. This is because the higher the amount of the at least one kind of oligonucleotide in the test sample, the higher the frequency of the signal.

[0085] The current change over time data may be used as raw data, or may be subjected to data processing for purposes such as obtaining statistical indicators from the current change over time data. The third step according to the present disclosure can apply, for example, a data processing method based on the central limit theorem described in the following (3-1) to (3-4). (3-1) Obtain a plurality of interval times from the current change over time data, (3-2) Perform processing based on the central limit theorem for the plurality of interval times to obtain a frequency distribution, (3-3) Identify the inclusion pattern derived from the at least one type of oligonucleotide based on the frequency distribution, (3-4) Determine the presence or absence of the at least one type of oligonucleotide in the test sample from the inclusion pattern.

[0086] By performing the third step according to the present disclosure by the method described in the above (3-1) to (3-4), even if there are variations in the interval time in the original current change data over time, a frequency distribution with a narrow distribution as a normal distribution can be obtained. As a result, for example, even if the difference in the interval time between different complexes such as complex HYB2 and complex HYB5 is small, attribution can be made with sufficient reliability based on the normal distribution with greatly reduced variations. That is, it becomes easier to determine the difference in the interval time attributed to each complex. Thereby, the presence or absence of each of one or more specific oligonucleotides in the measurement sample can be determined simply and accurately.

[0087] Hereinafter, each step of (3-1) to (3-4), which is an example of the third step according to the present disclosure, will be described in detail.

[0088] In the step of (3-1), a plurality of interval times are obtained from the current change data over time obtained in the second step.

[0089] The interval time represents "the time during which the current passing through the through-hole of the nanopore is inhibited due to the single-stranded part of the probe (i.e., the single-stranded part) formed by the dissociation of the specific oligonucleotide from the complex of the probe and the specific oligonucleotide entering the through-hole of the nanopore." More specifically, in the current change data over time, it represents the time from the current intensity passing through the through-hole of the nanopore until the current intensity decreases and then rises again.

[0090] The current intensity passing through the through-hole of the nanopore refers to the intensity of the current passing through the through-hole of the nanopore observed when a voltage is applied in a state where the probe and the specific oligonucleotide do not exist in the first solution, that is, in a state where the through-hole of the nanopore is not blocked.

[0091] The decrease in current intensity means that the single-stranded part of the probe (i.e., the single-stranded part) formed by the dissociation of the specific oligonucleotide from the complex of the probe and the specific oligonucleotide has invaded into the through-hole of the nanopore, thereby inhibiting the current passing through the through-hole of the nanopore and representing a state where the current intensity is lower than the current intensity passing through the through-hole of the nanopore.

[0092] As the state where the current intensity is lower than the current intensity passing through the through-hole of the nanopore, a set value as a judgment criterion may be provided, and when the current intensity is lower than the set value, it may be determined that the current intensity is in a decreased state. For example, in the current change data over time, the current value of the current passing through the through-hole of the nanopore is set as I 0 and a state where the current value is less than 75% of the set value compared with this current value I 0 may be determined as a state where the current intensity is decreased.

[0093] The fact that the current intensity rises again means that as the probe moves within the through-hole of the nanopore, the complex of the probe and the specific oligonucleotide is resolved, and the single probe passes to the second solution side, thereby observing again the current passing through the through-hole of the nanopore that has been inhibited. For example, in the current change data over time, a state where the current value rises again from a state where the current value is less than 75% of the set value compared with the current value I 0 to the current value I 0 may be determined as a state where the current intensity rises again.

[0094] The number of samples of the interval time obtained from the current change over time data, that is, the number of samples in the population, may be plural or more. If the frequency distribution obtained by the process based on the central limit theorem described later shows normality, it is not particularly limited. For example, the number of values of the interval time obtained from the current change over time data is preferably 50 or more, more preferably 100 or more, still more preferably 100 or more and 15,000 or less, and most preferably 100 or more and 1,000 or less. Note that the number of samples of the interval time corresponds to the number of raw data, and this is considered as the population in the present disclosure.

[0095] (3-2) In the step, a process based on the central limit theorem is performed on the plurality of interval times to obtain a frequency distribution.

[0096] The process based on the central limit theorem is a process including step A of randomly selecting n interval times from a plurality of interval times that are the population to form a sample population, step B of obtaining the sample mean of the sample population, and step C of repeating step A and step B. The process based on the central limit theorem may include other steps. Note that n represents an integer of 2 or more.

[0097] In step A, n interval times are randomly selected from a plurality of interval times that are the population to create a sample population. For example, n interval times can be randomly selected from the n interval times that are the population to create a sample population. Note that the n interval times randomly extracted from the population are allowed to be selected repeatedly from the population, and a number larger than the number of data in the population may be used as the sample population.

[0098] The number of samples n of the interval time randomly selected from a plurality of interval times that are the population may be 100 or more, 300 or more, 500 or more, 1,000 or more, or 10,000 or more.

[0099] When the number of samples \(n\) in the sample population is large (for example, 300 or more), the distribution of the sample mean obtained by averaging the sample population tends to be a normal distribution.

[0100] In step B, the sample mean of the sample population is obtained. The sample mean means the arithmetic mean value for the \(n\) samples randomly selected as described above.

[0101] In step C, steps A and B are repeated.

[0102] The number of repetitions of steps A and B is not particularly limited and may be appropriately set according to the dispersibility of the frequency distribution of the population mean in the population. For example, the number of repetitions of steps A and B is preferably 10,000 or more, more preferably 50,000 or more, and even more preferably 100,000 or more.

[0103] When the number of repetitions of steps A and B is large (for example, 10,000 or more), the frequency distribution tends to be a normal distribution. As a result, for example, even if the difference in the interval time between various complexes is small, a normal distribution with a greatly reduced variation can be obtained, enabling a highly reliable determination.

[0104] In the step of (3-3), based on the frequency distribution, the content pattern derived from the at least one kind of oligonucleotide is identified.

[0105] The frequency distribution may be a frequency distribution table in which the values of each sample mean are classes and the frequencies of the values of each sample mean are frequencies, or it may be a histogram of the frequency distribution table.

[0106] Identifying the content pattern derived from at least one kind of oligonucleotide based on the frequency distribution means identifying the frequency distribution obtained in the previous step as "the pattern of the frequency distribution specific to each specific oligonucleotide" or "the pattern of the frequency distribution specific to the complex of each specific oligonucleotide and the probe".

[0107] The third step may include a step of comparing the second frequency distribution obtained by performing the above (3-1) and (3-2) using, as the first frequency distribution, the frequency distribution and, as the test sample, a solution in which the presence or absence of each of at least one type of oligonucleotide is known.

[0108] When the third step includes a step of comparing the second frequency distribution and the first frequency distribution, the content patterns of various specific oligonucleotides can be obtained more clearly.

[0109] It is preferable to obtain the second frequency distribution for all types of complexes that can be formed between the specific oligonucleotide and the probe.

[0110] As a method for comparing the first frequency distribution and the second frequency distribution, for example, the confidence intervals of the respective sample means may be compared, or the confidence interval of the sample mean and the median may be compared.

[0111] The confidence interval of the sample mean is obtained by a t-test.

[0112] The confidence interval of the sample mean is preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more. When the confidence interval of the sample mean is 90% or more, it is considered that the presence or absence of at least one type of oligonucleotide in the test sample can be determined with higher accuracy.

[0113] As a method for comparing the first frequency distribution and the second frequency distribution, for example, the following mode may be adopted. First, from the second frequency distribution, for example, obtain the known information that 2250 ms to 2750 ms is the 95% confidence interval of the content pattern A of the complex in which all five types of specific oligonucleotides are hybridized to the probe. Next, when the median in the first frequency distribution is, for example, 2500 ms, since it is included in the confidence interval of the content pattern A, it is determined that it is the content pattern A.

[0114] In a normal distribution, the median and the mean value coincide. However, considering the case where the distribution is not completely a normal distribution, the mean value may be used instead of the median.

[0115] In the step (3-4), the presence or absence of each of the at least one type of oligonucleotide in the test sample is determined from the inclusion pattern.

[0116] When not all of the various specific oligonucleotides are included in the test sample, the frequency distribution unique to the probe alone may be identified as the pattern of the unique frequency distribution when not all of the various specific oligonucleotides are included in the first solution.

[0117] The third step according to the present disclosure may further include obtaining the content of each of the at least one type of oligonucleotide in the test sample based on the first frequency distribution.

[0118] In the third step according to the present disclosure, when obtaining the content of each of the at least one type of oligonucleotide in the test sample, quantitative information can be obtained for at least one type of oligonucleotide that is differentially expressed or not expressed in cancer or the like.

[0119] In the third step according to the present disclosure, the method for obtaining the content of each of the at least one type of oligonucleotide in the test sample is not particularly limited. For example, when applying data processing based on the central limit theorem in the third step, the third step may include a step of comparing the third frequency distribution obtained by performing the above (3-1) and (3-2) using a solution in which the content of the at least one type of oligonucleotide is known as the test sample, with the first frequency distribution.

[0120] Comparing the third frequency distribution containing quantitative information of at least one kind of oligonucleotide with the first frequency distribution enables more accurate acquisition of the quantitative information regarding at least one kind of oligonucleotide in the test sample.

[0121] In addition, when the concentration of at least one kind of oligonucleotide in the test sample increases, the concentration of the complex of at least one kind of oligonucleotide and the probe also increases. Therefore, the frequency distribution of the interval time (central value, arithmetic mean value, standard deviation, etc.) obtained by data processing based on the central limit theorem, the time when the nanopore is open, and the nanopore passing frequency of the probe change. The nanopore passing frequency of the probe is obtained from the formula [1 (molecule)] / [average time when the nanopore is open (ms)]. Based on this change, information about the amount of at least one kind of oligonucleotide can be obtained. Therefore, by obtaining the quantitative information regarding at least one kind of oligonucleotide through the above steps, the abundance of the specific oligonucleotide can be determined. Also, the concentration at which the detection limit of the specific oligonucleotide is reached can be estimated.

[0122] Note that instead of the second frequency distribution and / or the determination criteria based thereon, the frequency distribution and / or determination criteria obtained by simulation or the like may be used. Also, instead of the third frequency distribution and / or the determination criteria based thereon, the frequency distribution and / or determination criteria obtained by simulation or the like may be used.

[0123] (Probe) According to the present disclosure, there is provided a probe which is a DNA having the nucleotide sequence of SEQ ID NO: 1 "5'-GTCGAACGTTTTCGTTCGACCCTCATCTCGCCCGCAAAGACCCACCCTACCTGCACTGTAAGCACTTTTCCCACAAACCATTATGTGCTGCTACCCACTTATCAGGTTGTATTATAACCAACGGAACCACTAGTGACTTGCCCCCCCCCCCCCCCCCCCC-3'" (hereinafter referred to as "probe 1") or a probe which is a DNA having the nucleotide sequence of SEQ ID NO: 2 "5'-TCATCTCGCCCGCAAAGACCCACCCTACCTGCACTGTAAGCACTTTTCCCACAAACCATTATGTGCTGCTACCCACTTATCAGGTTGTATTATAACCAACGGAACCACTAGTGACTTGCCCCCCCCCCCCCCCCCCCC-3'" (hereinafter referred to as "probe 2").

[0124] Probes 1 and 2 have, on the 3'-terminal side, (n) x structure, that is, a polydeoxycytosine structure in which 20 cytosines are consecutive. Probe 1 has, on the 5'-terminal side, a hairpin structure 5'-GTCGAACGTTTTCGTTCGACCC-3'.

[0125] Probes 1 and 2 can be used, for example, as probes for determining the presence or absence of one or more of miR-193, miR-106a, miR-15a, miR-374, and miR-224 that are differentially expressed in cholangiocarcinoma.

[0126] Probes 1 and 2 have, in this order, nucleotide sequences that are completely complementary to miR-193, miR-106a, miR-15a, miR-374, and miR-224 from the 5'-terminal side in probes 1 and 2.

[0127] Probe 1 and Probe 2 have base sequences that are completely complementary to miR-193, miR-106a, miR-15a, miR-374, and miR-224. Therefore, when Probe 1 or Probe 2 is applied as a probe in miRNA measurement using nanopore analysis, the interval time can be suitably controlled.

[0128] In addition, Probe 1 and Probe 2 have base sequences that are complementary to miR-193, miR-106a, miR-15a, miR-374, and miR-224 in this order. Therefore, the free energy of the complex formed by Probe 1, or Probe 2, and miR-193, miR-106a, miR-15a, miR-374, and miR-224 is the lowest, and it is likely to exist stably. As a result, the variation in the interval time in the nanopore analysis technology of each complex is suppressed.

[0129] (Probe set) The probe according to the present disclosure may be a probe set. The probe set according to the present disclosure includes a probe that is a DNA having the base sequence of SEQ ID NO: 3 "5'-ACTACCTGCACTGTAAGACTATAAGCACTTTA-3'" (hereinafter referred to as "Probe 3-1") and a probe that is a DNA having the base sequence of SEQ ID NO: 4 "5'-CTACCTGCCACTTTG-3'" (hereinafter referred to as "Probe 3-2"). The probe set may further include other probes.

[0130] The probe set having Probe 3-1 and Probe 3-2 can be used, for example, as a probe set for determining the presence or absence of one or more miRNAs among miR-20a and miR-17-5p that are differentially expressed in small cell lung cancer.

[0131] In the probe set, probe 3-1 and probe 3-2 each have sequences complementary to miR-20a and miR-17-5p, respectively, in a state where they are split between the two probes. More specifically, when probe 3-1 and probe 3-2 coexist, there are sequences complementary to miR-20a and sequences complementary to miR-17-5a in a form that connects the two probes.

[0132] The complex formed only when all of miR-20a, miR-17-5p, probe 3-1, and probe 3-2 are present simultaneously, the complex formed by only one of miR-20a or miR-17-5p and the two probes, and the complex formed when only probe 3-1 and probe 3-2 are present can be clearly distinguished and measured in miRNA measurement using nanopore analysis.

[0133] ≪Method for Determining Whether a Test Subject Has a Specific Disease or Has a Risk of Developing a Specific Disease (Second Determination Method)≫ The method for determining whether a test subject of the present disclosure has a specific disease or has a risk of developing a specific disease (hereinafter, also referred to as the second determination method according to the present disclosure) is as follows. (1-1) A probe having a sequence complementary to one or more specific oligonucleotides that are differentially expressed in a diseased subject having a specific disease or an individual or tissue having a risk of developing a specific disease, and a sample derived from a test subject containing at least one oligonucleotide of the specific oligonucleotides are mixed in a test sample such that the concentration of the at least one oligonucleotide in the test sample is 1.0 aM to 1.0 pM, and the ratio of the concentration of the probe to the concentration of the at least one oligonucleotide is 1.0×10 4 :1 to 1.0×10 9 :1 to obtain a test sample. Or, (1-2) A probe having a sequence complementary to one or more specific oligonucleotides that are not differentially expressed in a subject having a specific disease or an individual or tissue at risk of developing a specific disease, and a sample derived from a test subject containing at least one of the specific oligonucleotides, wherein the concentration of the at least one oligonucleotide in the test sample is 1.0 aM to 1.0 pM, and the ratio of the concentration of the probe to the concentration of the at least one oligonucleotide is 1.0×10 4 :1 to 1.0×10 9 :1, to obtain a test sample, a first step; (2) Applying a voltage between the test sample as the first solution and a second solution separated from each other by a lipid bilayer having a nanopore, and measuring the current intensity of the current flowing between the first solution and the second solution over time to obtain current change data over time, a second step; (3) Based on the current change data over time, detecting the presence of the at least one oligonucleotide in the test sample, thereby determining whether the test subject has a specific disease or has a risk of developing a specific disease, a third step; comprising.

[0134] As described above, the second determination method according to the present disclosure can be applied in any of the cases of (1-1) and (1-2).

[0135] For example, when determining the presence of an oligonucleotide (hereinafter also referred to as miR-A) that is differentially expressed in a subject having a specific disease or an individual or tissue at risk of developing a specific disease but is not differentially expressed in a healthy subject (a healthy subject without a specific disease) or an individual or tissue not at risk of developing a specific disease, if it is determined that miR-A is present, it is suggested that the test subject has a specific disease or has a risk of developing a specific disease, and if it is determined that miR-A is not present, it is suggested that the test subject does not have a specific disease or does not have a risk of developing a specific disease. On the one hand, when determining the presence of an oligonucleotide (hereinafter also referred to as miR-B) that is not differentially expressed in an affected individual having a specific disease or an individual or its tissue having a risk of developing a specific disease, but is differentially expressed in a healthy individual (a healthy individual without the specific disease) or an individual or its tissue having no risk of developing a specific disease, if it is determined that miR-B is present, it is suggested that the test subject does not have the specific disease or has no risk of developing the specific disease, and if it is determined that miR-B is not present, it is suggested that the test subject has the specific disease or has a risk of developing the specific disease. Here, the above-mentioned tissue may be a pathological tissue in an affected individual having a specific disease or an individual having a risk of developing a specific disease, or a tissue of the same site in a healthy individual, or a tissue such as blood or other body fluids that is easy to sample.

[0136] Examples of the test subject according to the present disclosure include humans, non-human animals, etc. Examples of non-human animals include mammals excluding humans.

[0137] Examples of the sample obtained from the test subject include biological specimens. The biological specimen is not particularly limited and examples thereof include urine, blood, or saliva. Examples of the blood specimen include red blood cells, whole blood, serum, or plasma.

[0138] The sample obtained from the object to be inspected may be a liquid or a solid. For example, the undiluted solution of the sample obtained from the object to be inspected may be used as it is, or a diluted solution in which the sample is suspended, dispersed or dissolved in a medium may be used. When the sample obtained from the object to be inspected is a solid, for example, it is preferable to use a diluted solution in which the specimen is suspended, dispersed or dissolved in a solution medium as a liquid specimen. The solution medium is not particularly limited, and examples thereof include water or a buffer solution. For example, as the test sample as the first solution, when the sample is a liquid, the sample may be used as the test sample as it is, or a sample obtained by diluting the sample in a pH buffer solution or the like may be used as the test sample. The liquid specimen thus obtained can be used as the first solution or added to the first solution and used in the above-described determination method.

[0139] In the second determination method according to the present disclosure, the one or more specific oligonucleotides may be two or more specific oligonucleotides.

[0140] In the second determination method according to the present disclosure, the specific disease is not particularly limited, and may be, for example, cancer, a circulatory disease, a chronic inflammatory disease, a neurological disease, a metabolic disease, or the like.

[0141] Examples of the oligonucleotide that is differentially expressed in an affected individual having a specific disease, an individual having a risk of developing a specific disease, or a tissue thereof include microRNA.

[0142] Examples of the oligonucleotide that is not differentially expressed in an affected individual having a specific disease, an individual having a risk of developing a specific disease, or a tissue thereof include microRNA.

[0143] A probe having a sequence complementary to one or more specific oligonucleotides differentially expressed in a subject having a specific disease, an individual having a risk of developing a specific disease, or their tissues means, for example, a probe having a sequence complementary to one or more oligonucleotides differentially expressed in a subject having a specific disease, an individual having a risk of developing a specific disease, or their tissues as compared to a healthy subject without the specific disease, an individual without a risk of developing the specific disease, or their tissues. Here, in a healthy subject without the specific disease, an individual without a risk of developing the specific disease, or their tissues, the expression of the oligonucleotide may be sufficiently low, and may be complete non-expression or even a small amount of expression. Also, when the quantitative information of the oligonucleotide is used for determination in the second determination method, the expression of the oligonucleotide in a healthy subject without the specific disease, an individual without a risk of developing the specific disease, or their tissues may be significantly lower than the expression of the oligonucleotide in a subject having the specific disease, an individual having a risk of developing the specific disease, or their tissues.

[0144] A probe having a sequence complementary to one or more specific oligonucleotides that are not differentially expressed in a subject having a specific disease, an individual having a risk of developing a specific disease, or their tissues means, for example, a probe having a sequence complementary to one or more oligonucleotides that are not differentially expressed in a subject having a specific disease, an individual having a risk of developing a specific disease, or their tissues as compared to a healthy subject without the specific disease, an individual without the risk of developing the specific disease, or their tissues. Here, in a subject having a specific disease, an individual having a risk of developing a specific disease, or their tissues, it is sufficient that the expression of the oligonucleotide is sufficiently low, and it may be complete non-expression or even a small amount of expression. Also, when the quantitative information of the oligonucleotide is used for the determination in the second determination method, the expression of the oligonucleotide in a subject having a specific disease, an individual having a risk of developing a specific disease, or their tissues should be significantly lower than the expression of the oligonucleotide in a healthy subject without the specific disease, an individual without the risk of developing the specific disease, or their tissues.

[0145] The probe in the second determination method according to the present disclosure may have the same configuration as the probe in the first determination method described above, except that it has a sequence complementary to one or more specific oligonucleotides that are differentially expressed or not expressed in a subject having a specific disease, an individual having a risk of developing a specific disease, or their tissues.

[0146] In the second determination method according to the present disclosure, the first step, the second step, and the third step may be the same steps as the first step, the second step, and the third step in the aforementioned first determination method, except that the test sample is taken as "a sample derived from the object to be inspected" and the probe is taken as "a probe having a sequence complementary to one or more specific oligonucleotides that are differentially expressed or not expressed in an individual having a specific disease or an individual at risk of developing a specific disease or in their tissues". However, in the third step of the second determination method, it also includes determining whether the object to be inspected has a specific disease or has a risk of developing a specific disease based on the presence information (and optionally quantitative information) of the at least one oligonucleotide. Specifically, reference can be made to the description in the case of miR-A or miR-B described above.

[0147] In the second determination method according to the present disclosure, when applying data processing based on, for example, the central limit theorem in the third step, in step (3-3), the frequency distribution A obtained using a sample derived from the object to be inspected is compared with the frequency distribution B obtained by performing the above (1) to (3-2) using a sample derived from a healthy person not suffering from a specific disease or an individual not at risk of developing a specific disease instead of the sample, and / or the frequency distribution C obtained by performing the above (1) to (3-2) using a sample derived from an individual suffering from a specific disease or an individual at risk of developing a specific disease, thereby determining whether the object to be inspected has a specific disease or has a risk of developing a specific disease.

[0148] A healthy individual who does not suffer from a specific disease or an individual who does not have a risk of suffering from a specific disease means an individual who is known not to suffer from a specific disease or not to have a risk of suffering from a specific disease. In a healthy individual who does not suffer from a specific disease or an individual or their tissue that does not have a risk of suffering from a specific disease, one or more specific oligonucleotides that are differentially expressed in an affected individual with a specific disease or an individual or their tissue that has a risk of suffering from a specific disease are not expressed, and one or more specific oligonucleotides that are not differentially expressed in an affected individual with a specific disease or an individual or their tissue that has a risk of suffering from a specific disease are expressed. As used herein, "not expressed" is not limited to complete non-expression as described above.

[0149] An affected individual who suffers from a specific disease or an individual who has a risk of suffering from a specific disease means an individual who is known to suffer from a specific disease or to have a risk of suffering from a specific disease. In an affected individual who suffers from a specific disease or an individual or their tissue that has a risk of suffering from a specific disease, one or more specific oligonucleotides that are differentially expressed in an affected individual with a specific disease or an individual or their tissue that has a risk of suffering from a specific disease are expressed, and one or more specific oligonucleotides that are not differentially expressed in an affected individual with a specific disease or an individual or their tissue that has a risk of suffering from a specific disease are not expressed. As used herein, "not expressed" is not limited to complete non-expression as described above.

[0150] In the third step, when applying data processing based on, for example, the central limit theorem, comparing the frequency distribution A obtained using samples derived from the subject to be tested with the frequency distribution B obtained using samples derived from healthy individuals who do not suffer from a specific disease or individuals who do not have a risk of suffering from a specific disease may include comparing the confidence intervals of the sample means in the frequency distributions of each sample, may include comparing the confidence interval of the sample mean with the median, or may include comparing the medians. For example, from the perspective of more accurately determining whether the subject to be tested suffers from a specific disease or has a risk of suffering from a specific disease, comparing the frequency distribution A and the frequency distribution B preferably includes comparing the confidence intervals of the sample means in the frequency distributions of each sample.

[0151] The confidence interval of each frequency distribution is obtained by a t-test.

[0152] As the confidence interval in each frequency distribution, it is preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 99% or more.

[0153] If the confidence interval in each frequency distribution is 80% or more, it is considered that whether the subject to be tested suffers from a specific disease or has a risk of suffering from a specific disease can be determined more accurately.

[0154] The frequency distribution B and the frequency distribution C may be frequency distributions obtained by actual measurement or may be frequency distributions predicted by referring to papers or the like.

[0155] When determining samples derived from a plurality of different test subjects, the step of obtaining the frequency distribution B using samples derived from healthy individuals not suffering from a specific disease or individuals having no risk of suffering from the specific disease does not necessarily need to be performed every time. More specifically, the frequency distribution B may be obtained in advance using samples derived from healthy individuals not suffering from a specific disease or individuals having no risk of suffering from the specific disease, and this frequency distribution B may be compared with and determined against samples derived from a plurality of different test subjects.

[0156] When determining samples derived from a plurality of different test subjects, the step of obtaining the frequency distribution C using samples derived from affected individuals suffering from a specific disease or individuals having a risk of suffering from the specific disease does not necessarily need to be performed every time. More specifically, the frequency distribution C may be obtained in advance using samples derived from affected individuals suffering from a specific disease or individuals having a risk of suffering from the specific disease, and this frequency distribution C may be compared with and determined against samples derived from a plurality of different test subjects.

[0157] The frequency distribution A obtained using the sample derived from the test subject may be appropriately corrected according to the age, physiological parameters, etc. of the test subject.

[0158] The determination criteria will be described below.

[0159] As a determination criterion for determining that the test subject does not suffer from a specific disease, the area of the histogram of the confidence interval in the frequency distribution A obtained using the sample derived from the test subject is compared with the area of the histogram of the confidence interval in the frequency distribution B obtained using the sample derived from a healthy individual not suffering from the specific disease. If the areas of both overlap by 50% or more (more preferably 60% or more), it may be determined that the test subject does not suffer from the specific disease.

[0160] Further, compare the confidence interval of the frequency distribution A obtained using a sample derived from the test subject with the median of the frequency distribution B obtained using a sample derived from a healthy subject who does not have the specific disease. Even when the median is included in the confidence interval, it may be determined that the test subject does not have the specific disease.

[0161] In addition, compare the area of the histogram of the confidence interval in the frequency distribution C obtained using a sample derived from a patient suffering from the specific disease with the area of the histogram of the confidence interval in the frequency distribution A. When the areas of the two do not overlap, it may be determined that the test subject does not have the specific disease. At this time, further compare the area of the histogram of the confidence interval in the frequency distribution B obtained using a sample derived from a healthy subject who does not have the specific disease with the area of the histogram of the confidence interval in the frequency distribution A. After confirming that the areas of the two overlap by 50% or more (more preferably 60% or more), it can also be determined that the test subject does not have the specific disease. Alternatively, compare the confidence interval of the frequency distribution A obtained using a sample derived from the test subject with the median of the frequency distribution B obtained using a sample derived from a healthy subject who does not have the specific disease. After confirming that the median is included in the confidence interval, it can also be determined that the test subject does not have the specific disease.

[0162] On the other hand, as a criterion for determining that the test subject has the specific disease, compare the frequency distribution A obtained using a sample derived from the test subject with the frequency distribution C obtained using a sample derived from a patient suffering from the specific disease. When the areas of the histograms of the confidence intervals in the confidence intervals of the two frequency distributions overlap by 50% or more (more preferably 60% or more), it may be determined that the test subject has the specific disease.

[0163] Further, compare the confidence interval of the frequency distribution A obtained using a sample derived from the subject to be tested with the median of the frequency distribution C obtained using a sample derived from a patient suffering from a specific disease. Even when the median is included in the confidence interval, it may be determined that the subject to be tested is suffering from the specific disease.

[0164] In addition, compare the area of the histogram of the confidence interval in the frequency distribution B obtained using a sample derived from a healthy person not suffering from the specific disease with the area of the histogram of the confidence interval in the frequency distribution A. When the areas of both do not overlap, it may be determined that the subject to be tested is suffering from the specific disease. At this time, further compare the area of the histogram of the confidence interval in the frequency distribution C obtained using a sample derived from a patient suffering from the specific disease with the area of the histogram of the confidence interval in the frequency distribution A. After confirming that the areas of both overlap by 50% or more (more preferably 60% or more), it can also be determined that the subject to be tested is suffering from the specific disease. Or, compare the confidence interval of the frequency distribution A obtained using a sample derived from the subject to be tested with the median of the frequency distribution C obtained using a sample derived from a patient suffering from the specific disease. After confirming that the median is included in the confidence interval, it can also be determined that the subject to be tested is suffering from the specific disease.

[0165] The determination may be made, for example, specifically, in the following manner. First, obtain known information from the frequency distribution B that, for example, 900 ms to 1200 ms is the 95% confidence interval in the frequency distribution of samples derived from healthy persons not suffering from the specific disease. Next, obtain information from the frequency distribution A that, for example, 2250 ms to 2750 ms is the 95% confidence interval in the frequency distribution of samples derived from the subject to be tested. Then, when comparing the 95% confidence intervals in these frequency distributions A and B, the 95% confidence intervals of both do not overlap 100%. Therefore, it is determined that the subject to be tested is suffering from the specific disease.

[0166] Note that instead of the frequency distribution B and / or the determination criteria based thereon, the frequency distribution and / or determination criteria obtained by simulation or the like may be used. Further, instead of the frequency distribution C and / or the determination criteria based thereon, the frequency distribution and / or determination criteria obtained by simulation or the like may be used.

[0167] Note that in a normal distribution, the median and the mean value coincide. However, considering the case where the distribution is not completely normal, the mean value may be used instead of the median. Further, the determination may be a definite determination or may not be a definite determination. In the case where the determination is not a definite determination (i.e., in the case of a preliminary determination), further tests may be performed to make a definite determination for the subject preliminarily determined to have the specific disease in the preliminary determination. According to the second determination method according to the present disclosure, since it is possible to determine the presence or absence of a trace amount of a specific oligonucleotide, it is possible to make a determination even in the early stage of the progression of the disease (for example, stage 0). Since the symptoms of the disease often do not appear at such an early stage, the accuracy of the determination may be further improved by performing additional tests. Further, when determining the risk of having the disease, a high-risk group consisting of individuals having a risk of developing a specific disease is defined based on a known method, and a low-risk group consisting of individuals having no or a low risk of developing the specific disease is defined. An individual belonging to the high-risk group may be used instead of a patient having the specific disease, and an individual belonging to the low-risk group may be used instead of a healthy person not having the specific disease. For example, as a determination criterion for determining that the test subject has no risk of developing a specific disease, the area of the histogram of the confidence interval in the frequency distribution A obtained using a sample derived from the test subject is compared with the area of the histogram of the confidence interval in the frequency distribution B obtained using a sample derived from an individual belonging to a low-risk group having no risk of developing the specific disease. If the areas of both do not overlap, it may be determined that the test subject has a risk of developing the specific disease. Other exemplary methods can be applied in the same manner. In general, the high-risk group and the low-risk group can be defined by observing whether a specific disease has occurred after performing time-course monitoring (e.g., for 3 years, 10 years, lifetime, etc.).

[0168] (Cholangiocarcinoma) In the second determination method according to the present disclosure, the specific disease may be cholangiocarcinoma.

[0169] Examples of oligonucleotides that are differentially expressed in patients with cholangiocarcinoma or their tissues include miR-374, miR-15a, miR-224, miR-106a, miR-193, etc.

[0170] Examples of oligonucleotides that are not differentially expressed in patients with cholangiocarcinoma or their tissues include miR-20a, miR-17-5p, etc.

[0171] (Small cell lung cancer) In the second determination method according to the present disclosure, the specific disease may be small cell lung cancer.

[0172] Examples of the test subject and the sample obtained from the test subject include the same test subjects as those exemplified in the method for determining the possibility of cholangiocarcinoma.

[0173] Examples of specific oligonucleotides that are differentially expressed in patients with small cell lung cancer or their tissues include miR-20a, miR-17-5p, etc.

[0174] Examples of specific oligonucleotides that are not differentially expressed in patients with small cell lung cancer or their tissues include miR-193, miR-374, etc.

Example

[0175] Hereinafter, the present disclosure will be described more specifically by way of examples. However, the present disclosure is not limited to the following examples as long as it does not exceed the gist thereof. Unless otherwise specified, "parts" are based on mass. The same applies to "%", which is also based on mass.

[0176] - Preparation of Materials - The following materials were used.

[0177] (Preparation of Specific Oligonucleotides (miRNA)) Each miRNA having the nucleotide sequence shown below was synthesized using high-performance liquid chromatography grade (manufactured by FastMac Co., Ltd.) and used as a 100 nmol / ml solution.

[0178] miR-374 SEQ ID NO: 5: 5'-UUAUAAUACAACCUGAUAAGUG-3' miR-15a SEQ ID NO: 6: 5'-UAGCAGCACAUAAUGGUUUGUG-3' miR-224 SEQ ID NO: 7: 5'-CAAGUCACUAGUGGUUCCGUU-3' miR-106a SEQ ID NO: 8: 5'-AAAAGUGCUUACAGUGCAGGUAG-3' miR-193 SEQ ID NO: 9: 5'-UGGGUCUUUGCGGGCGAGAUGA-3'

[0179] (Buffer Solution) A buffer solution of 1.0 M KCl and 10 mM MOPS, pH 7.0 was used.

[0180] (Preparation of Probe 1 and Probe 2) Probes 1 and 2 were designed for their base sequences using NUPACK of Caltech (California Institute of Technology) which enables the design of nucleic acid bases and the like. The base sequences of the probes were designed to be completely complementary sequences from the 5'-end side in Probes 1 and 2 in this order with respect to five specific oligonucleotides miR-193, miR-106a, miR-15a, miR-374, and miR-224, which are known to be differentially expressed in cholangiocarcinoma. Furthermore, Probe 1 was designed to have a hairpin structure on the 5'-end side and a polydeoxycytosine structure on the 3'-end side, and Probe 2 was designed to have a polydeoxycytosine structure on the 3'-end side. Next, oligonucleotides having the designed base sequences were synthesized respectively by high performance liquid chromatography grade (manufactured by Fastmac Co., Ltd.), and these were used as Probe 1 or Probe 2. Probe 1 has the base sequence of SEQ ID NO: 1 "5'-GTCGAACGTTTTCGTTCGACCCTCATCTCGCCCGCAAAGACCCACCCTACCTGCACTGTAAGCACTTTTCCCACAAACCATTATGTGCTGCTACCCACTTATCAGGTTGTATTATAACCAACGGAACCACTAGTGACTTGCCCCCCCCCCCCCCCCCCCC-3'". Probe 2 has the base sequence of SEQ ID NO: 2 "5'-TCATCTCGCCCGCAAAGACCCACCCTACCTGCACTGTAAGCACTTTTCCCACAAACCATTATGTGCTGCTACCCACTTATCAGGTTGTATTATAACCAACGGAACCACTAGTGACTTGCCCCCCCCCCCCCCCCCCCC-3'".

[0181] [Example 1] -Determination of five miRNAs differentially expressed in cholangiocarcinoma- (First step) Blood samples were collected from 6 patients with cholangiocarcinoma and 6 healthy subjects (non-cholangiocarcinoma patients). Plasma was obtained from each of the blood samples by centrifugation. A solution containing miRNA was extracted from the plasma. In addition, a solution was prepared by adding 1 μl of synthetic RNA (cel-miR39-3p; SEQ ID NO: 10: 5'-ucaccggguguaaaucagcuug-3'; manufactured by Fastenakel Co., Ltd.) as a control to 299 μl of the plasma, and miRNA was extracted from this solution. Specifically, miRNA was extracted using a commercially available RNA purification kit, NucleoSpin (registered trademark) miRNA Plasma (Takara Bio Inc.). According to the kit instructions provided by the manufacturer, precipitation and removal of proteins in the plasma, binding of miRNA to the column, DNase treatment, washing, and elution of miRNA were performed. By the above operations, miRNA solutions were obtained.

[0182] For each miRNA solution extracted from the plasma, a sample of 9.9 μl containing 4.8 μl of the miRNA solution, 0.1 μl of probe 1 (50 μM), and 5 μl of KCl solution and MOPS solution was prepared. At this time, the sample was prepared so that the probe concentration was 500 nM, the KCl concentration was 1 M, and the MOPS concentration was 10 mM in the final (first solution) test sample.

[0183] (Annealing treatment step) The 9.9 μl samples were heated at 95°C for 5 minutes and then subjected to annealing treatment by gradually cooling to room temperature (25°C).

[0184] (Second step) A lipid bilayer with a nanopore was provided on one well using dipalmitoyl-sn-glycero-3-phosphocholine (DPhPC, Avanti Polar Lipids, Inc.) as the lipid, and the first solution and the second solution were separated within the well by the lipid bilayer. For the detailed method of separating the first solution and the second solution within the well, reference can be made to the method described in M. OHARA, Y. SEKIYA, R. KAWANO, Hairpin DNA Unzipping Analysis Using a Biological Nanopore Array, Electrochemistry, 84(5), 338 - 341 (2016). To each of the 9.9 μl of the annealed samples, 0.1 μl of the nanopore was added to make a total of 10 μl, and 4.7 μl of the sample (test sample) was used as the first solution. The second solution was 4.7 μl of 1 M KCl and 10 mM MOPS (pH 7.0). The nanopore used was the wild-type αHL, a monomeric polypeptide isolated from Staphylococcus aureus (Sigma-Aldrich, St. Louis, MO and USA, List Biological Laboratories, Campbell, CA, USA), and the concentration in the first solution was 30 nM. Next, a constant voltage of +200 mV was applied from the first solution to the second solution direction, and the current change data over time was acquired using Clampex 9.0 (Molecular Devices) equipped with a Digidata 1440A analog-to-digital converter (Molecular Devices). The current applied was recorded using an Axopatch 200B Amplifier (Molecular Devices, San Jose, CA, USA). A schematic cross-sectional view of the nanopore analysis technique showing the application of voltage using the lipid bilayer with a nanopore, the first solution (cis), and the second solution (trans) is shown in Figure 4.

[0185] (Third step) Based on the current-time change data for each test sample obtained in the second step, a frequency distribution was obtained. First, 300 interval times were acquired from the current-time change data and used as the population. Next, 300 interval times were randomly selected from the 300 interval times and used as the sample population. Then, the arithmetic mean of this sample population was calculated and used as the sample mean. The process of creating this sample population and calculating the sample mean was repeated 2 16 times to obtain the frequency distribution. In the present disclosure, the population is used in the sense of measurement data before statistical processing. Based on the current-time change data for each test sample obtained in the second step, the 2 16 histograms of the obtained sample means were created with the interval time (Unzipping time [ms] in the figure) shown in logarithmic scale (Figure 5). In the histogram of each composite, the frequency (number of times, Frequency in the figure) on the vertical axis represents the value normalized at the median of each normal distribution. Also, in the figure, Peak indicates the median of the histogram. The terms and abbreviations shown in the figure have the same meaning in the following figures.

[0186] The results of the histograms obtained from 6 patients with bile duct cancer were averaged (Figure 6). Furthermore, the results of the histograms obtained from 6 healthy subjects (non-bile duct cancer patients) were also averaged (Figure 6). The peak for the bile duct cancer patients was 1487 ms, while the peak for the healthy subjects was 856 ms. Specifically, when comparing the 90% confidence intervals in each histogram, these confidence intervals do not overlap. Therefore, it can be said that the histograms of various composites could be compared and distinguished from each other with sufficient confidence. Test samples based on samples obtained from cholangiocarcinoma patients contain specific oligonucleotides that are differentially expressed in cholangiocarcinoma patients. Therefore, it can be said that it is difficult for the probe to pass through the nanopore and the interval time is long. On the other hand, in test samples based on samples obtained from healthy individuals, since the concentration of specific oligonucleotides that are differentially expressed in cholangiocarcinoma patients is low, it can be said that the probe easily passes through the nanopore and the interval time is short.

[0187] [Example 2] -Measurement of the concentrations of five miRNAs in plasma samples- miRNA solutions of five cholangiocarcinoma patients (sample Nos. T1 to T5) and five healthy individuals (sample Nos. N1 to N5) were prepared. The miRNA solutions of five cholangiocarcinoma patients and five healthy individuals were prepared by selecting five miRNA solutions each from the miRNA solutions of six cholangiocarcinoma patients and six healthy individuals prepared in [Example 1].

[0188] Using the miRNA solution, a reverse transcription reaction of miRNA was performed. Specifically, a commercially available miRNA quantification kit Mir-X miRNA qRT-PCR TB Green Kit (Takara Bio Inc.) was used. According to the kit instructions provided by the manufacturer, polyadenylation and SMART (registered trademark) MMLV Reverse Transcriptase as a reverse transcriptase were used to synthesize cDNA under the conditions of 37°C for 1 hour and 85°C for 5 minutes.

[0189] PCR was performed for each miRNA in the synthesized cDNA. Specifically, a commercially available miRNA quantification kit Mir-X miRNA qRT-PCR TB Green Kit (Takara Bio Inc.) was used. According to the kit instructions provided by the manufacturer, a PCR reaction solution was prepared as follows. After heat denaturation at 95°C for 10 seconds, qPCR was performed under the conditions of 95°C for 5 seconds and 60°C for 25 seconds for 40 cycles. The melting curve analysis temperature was set at 95°C for 60 seconds, 55°C for 30 seconds, and 95°C for 30 seconds. ddH 2 O 9.5μl TB Green Advantage Premix(2x) 12.5 μl miRNA-specific primer(10 μM) 0.5 μl mRQ 3’Primer(10 μM) 0.5 μl cDNA 2.0 μl Total 25.0 μl

[0190] Note: As the reverse primer, mRQ 3’Primer included in the above kit was used. As the forward primer, miRNA-specific primer, for each miRNA, oligonucleotides with the base sequences indicated by the following SEQ ID Nos. were used. The forward primer used was manufactured by Fastmacc Co., Ltd. miR-374: Forward primer SEQ ID No. 11: 5'-TTATAATACAACCTGATAAGTG-3' miR-15a: Forward primer SEQ ID No. 12: 5'-TAGCAGCACATAATGGTTTGTG-3' miR-224: Forward primer SEQ ID No. 13: 5'-CAAGTCACTAGTGGTTCCGTT-3' miR-106a: Forward primer SEQ ID No. 14: 5'-AAAAGTGCTTACAGTGCAGGTAG-3' miR-193: Forward primer SEQ ID No. 15: 5'-TGGGTCTTTGCGGGCGAGATGA-3' cel-miR-39-3p: Forward primer SEQ ID No. 16: 5'-TCACCGGGTGTAAATCAGCTTG-3'

[0191] A graph showing the concentration of each miRNA detected in the plasma sample is shown in Fig. 7. In the figure, for five patients with cholangiocarcinoma (sample Nos. T1 to T5) and five healthy subjects (sample Nos. N1 to N5), from left to right, the concentrations (pM) of miR-193, miR-106a, miR-15a, miR-224, and miR-374 are shown respectively. The concentration of each miRNA in the figure represents the value converted to the concentration in the plasma sample. The concentration of each miRNA ranged from a high concentration of approximately 100 pM to a very low concentration of 10 aM (miR-15a). In the vertical axis of Fig. 7, for example, the notation "1.E-02" indicates that it is the miRNA concentration of 1.0×10 -2 pM.

[0192] For five patients with cholangiocarcinoma and five healthy subjects, the average value of the concentration of each miRNA was calculated (Fig. 8). The concentration of each miRNA in the figure represents the value converted to the concentration in the plasma sample. In patients with cholangiocarcinoma, the average value of the concentration of each miRNA was 10.8 pM for miR-193, 5.71 fM for miR-106a, 1.19 fM for miR-15a, 580 aM for miR-224, and 133 fM for miR-374. In healthy subjects, the average value of the concentration of each miRNA was 3.42 pM for miR-193, 5.11 fM for miR-106a, 1.16 fM for miR-15a, 28.7 aM for miR-224, and 3.73 fM for miR-374. That is, in Example 1, it was found that the difference in the interval time between patients with cholangiocarcinoma and healthy subjects, which reflected the difference in the miRNA concentration at the aM to pM level, could be detected for each miRNA in patients with cholangiocarcinoma and healthy subjects. In the vertical axis of Fig. 8, for example, the notation "1.E-02" indicates that it is the miRNA concentration of 1.0×10 -2 pM.

[0193] [Example 3] -Determination for miRNAs at very low concentrations- (First step) Sample 1 was prepared so that the probe 2 in the final test sample 1 (as the first solution) would be 500 nM. Sample 2 was prepared so that the probe 2 in the final test sample 2 (as the first solution) would be 500 nM and miR-15a would be 100 fM. Sample 3 was prepared so that the probe 2 in the final test sample 3 (as the first solution) would be 500 nM and miR-15a and miR-193 would each be 100 fM. Sample 4 was prepared so that the probe 2 in the final test sample 4 (as the first solution) would be 500 nM and miR-15a, miR-193, and miR-224 would each be 100 fM. Sample 5 was prepared so that the probe 2 in the final test sample 5 (as the first solution) would be 500 nM and miR-15a, miR-193, miR-224, and miR-106a would each be 100 fM. Sample 6 was prepared so that the probe 2 in the final test sample 6 (as the first solution) would be 500 nM and miR-15a, miR-193, miR-224, miR-106a, and miR-374 would each be 100 fM.

[0194] (Annealing process) Samples 1 to 6 were each heated at 95 °C for 5 minutes and then gradually cooled to room temperature (25 °C) for annealing treatment.

[0195] (Second step) For Samples 1 to 6, in the same manner as in [Example 1], the first solution (Samples 1 to 6 as test samples 1 to 6, respectively) and the second solution were separated in the well. Next, a constant voltage of +200 mV was applied from the first solution to the second solution, and the current-time change data was acquired using Clampex 9.0 (Molecular Devices) equipped with a Digidata 1440A analog-to-digital converter (Molecular Devices). The current applied using an Axopatch 200B Amplifier (Molecular Devices, San Jose, CA, USA) was recorded.

[0196] (Third step) Based on the current-time change data for test samples 1 to 6 obtained in the second step, 2 16 histograms with logarithmic display of the interval time were created for the frequency distributions of the two sample averages obtained in the same manner as in Example 1 (Fig. 9). In the histograms, the frequency (number of times) on the vertical axis indicates the value normalized at the median of each normal distribution. In the figure, dgDNAwoh indicates probe 2. From this result, it became clear that the presence of miRNA could be detected using probe 2 for miRNA at an extremely low concentration of 100 fM.

[0197] (Correlation analysis between interval time and free energy) Using NUPACK simulation (http: / / www.nupack.org), the free energies of the probe and miRNA in test samples 1 to 6 were calculated. With the peak (ms) of the interval time obtained above on the vertical axis and the calculated free energy (Free energy; kJ / mol) on the horizontal axis, the results of test samples 1 to 6 were plotted respectively (Fig. 10). In the figure, circles 1 to 6 indicate the plots and their error bars in test samples 1 to 6 respectively.

[0198] Since NUPACK simulation cannot determine the free energy when five or more types of miRNAs bind, the free energy for test sample 6 was calculated by the following method. First, the free energy A (five patterns) when probe 2 binds to four types of miRNAs selected from the group consisting of miR-15a, miR-193, miR-224, miR-106a, and miR-374 was calculated. Next, the free energy B (one pattern of free energy B for each of the five patterns of free energy A) when the remaining one type of miRNA not selected from the group binds to probe 2 was calculated. The free energy B corresponding to each free energy A was summed up, and the average value of the five patterns of "free energy A + free energy B" was taken as the free energy in test sample 6.

[0199] When an approximate line was obtained based on the plot of the free energy between the probe and miRNA, the linear equation y = -1.02x + 162.80 was obtained, and the coefficient of determination R 2 was 1.00, and a negative correlation was confirmed between the interval time and the free energy. That is, the more miRNAs bound to the probe, the closer the free energy approached a negative value, and the complex was formed stably.

[0200] [Example 4] -Determination depending on the concentration of miRNA- (First step) Sample 7 was prepared so that the concentration of probe 2 in the final test sample 7 (as the first solution) was 500 nM. Sample 7 was used as a negative control in the following tests regarding miR-15a and miR-193. Sample 8 was prepared so that the concentration of probe 2 in the final test sample 8 (as the first solution) was 500 nM and the concentration of miR-15a was 1.0 fM. Sample 9 was prepared so that the concentration of probe 2 in the final test sample 9 (as the first solution) was 500 nM and the concentration of miR-15a was 10 fM. Sample 10 was prepared such that the probe 2 in the final test sample 10 (as the first solution) was 500 nM and miR-15a was 50 fM. Sample 11 was prepared such that the probe 2 in the final test sample 11 (as the first solution) was 500 nM and miR-15a was 100 fM. Sample 12 was prepared such that the probe 2 in the final test sample 12 (as the first solution) was 500 nM and miR-15a was 200 fM. Sample 13 was prepared such that the probe 2 in the final test sample 13 (as the first solution) was 500 nM and miR-193 was 1.0 fM. Sample 14 was prepared such that the probe 2 in the final test sample 14 (as the first solution) was 500 nM and miR-193 was 10 fM. Sample 15 was prepared such that the probe 2 in the final test sample 15 (as the first solution) was 500 nM and miR-193 was 50 fM.

[0201] (Annealing process) The samples 7 to 15 were each heated at 95 °C for 5 minutes and then gradually cooled to room temperature (25 °C) for annealing.

[0202] (Second step) For the samples 7 to 15, in the same manner as in [Example 1], the first solution (the samples 7 to 15 as the test samples 7 to 15 respectively) and the second solution were separated in the well. Next, a constant voltage of +200 mV was applied from the first solution toward the second solution, and the current change data over time were acquired using Clampex 9.0 (Molecular Devices) equipped with a Digidata 1440A analog-to-digital converter (Molecular Devices). The current applied was recorded using an Axopatch 200B Amplifier (Molecular Devices, San Jose, CA, USA).

[0203] (Third step) Based on the current change data over time for test samples 7 to 15 obtained in the second step, 2 16 histograms with logarithmic display of the interval time were created for the frequency distributions of the two sample means (Figure 11). In the histograms of each complex, the frequency (number of times) on the vertical axis indicates the value normalized at the median of each normal distribution. In the figure, dgDNAwoh indicates probe 2.

[0204] Taking the peak (ms) of each interval time obtained for test samples 7 to 15 on the vertical axis and the concentration (fM) of miRNA in test samples 7 to 15 on the horizontal axis, the results of test samples 7 to 15 were plotted respectively (Figure 12). When an approximate line was obtained from the plot, for miR-15a, the linear equation y = 1.21x + 254.76 was obtained, and the coefficient of determination R 2 was 0.94, and a positive correlation was confirmed between the interval time and the concentration of miRNA (black circles and approximate dotted line in the figure). For miR-193 as well, the linear equation y = 6.73x + 274.82 was obtained, and the coefficient of determination R 2 was 0.78, and a positive correlation was confirmed between the interval time and the concentration of miRNA (white circles and approximate dotted line in the figure). That is, even for miRNA at an extremely low concentration at the fM level, miRNA could be accurately detected by using the method described above.

[0205] [Example 5] -Nanopore Passage Dependent on Probe Concentration- (First step) Sample 16 was prepared such that probe 2 in test sample 16 was 500 nM and miR-15a was 1.0 fM. Alternatively, sample 17 was prepared such that probe 2 in test sample 17 was 500 pM and miR-15a was 1.0 fM.

[0206] (Annealing process step) Samples 16 to 17 were each heated at 95°C for 5 minutes and then gradually cooled to room temperature (25°C) for annealing treatment.

[0207] (Second step) For samples 16 to 17, in the same manner as in [Example 1], the first solution (samples 16 to 17 as test samples 16 to 17, respectively) and the second solution were separated within the well. Next, a constant voltage of +200 mV was applied from the first solution to the second solution direction, and the current change data over time was acquired using Clampex 9.0 (Molecular Devices) equipped with a Digidata 1440A analog-to-digital converter (Molecular Devices). The current applied using an Axopatch 200B Amplifier (Molecular Devices, San Jose, CA, USA) was recorded.

[0208] (Third step) Based on the current-time change data for test samples 1 to 2 obtained in the second step, the time during which the nanopore was open (open time (ms)) was determined (Figure 13). In the example of the current-time change data on the left side of Figure 13, the vertical axis represents the current intensity, and the horizontal axis represents the measurement time. As shown in the example of the current-time change data on the left side of Figure 13, the "time during which the nanopore was open" was defined as the time until the current intensity exceeded 0% and was less than 80% assuming that the current intensity when the through-hole of the nanopore was open (open) was 0% and the current intensity when the through-hole of the nanopore was closed (Base) was 100%. That is, the time during which the nanopore was open (open time (ms)) is the time during which the probe did not penetrate into the through-hole of the nanopore. The graph (box-and-whisker plot) on the right side of Figure 13 shows the time during which the nanopore was open when the concentration of the probe was changed. The box chart of the box-and-whisker plot shows the range corresponding to the first quartile to the third quartile of the time during which the nanopore was open, and the center line of the box chart shows the median (second quartile). In the figure, the error bars show the data range excluding outliers, the × marks show the values of 1% and 99% of the data, and the □ marks show the average value.

[0209] When the probe concentration (diagnostic DNA concentration in the figure) was 500 nM, the time during which the nanopore was open was about 0 ms. On the other hand, when the probe concentration was 500 pM, the median of the time during which the nanopore was open was about 1000 ms.

[0210] (Calculation of nanopore passing frequency) The nanopore passing frequency was calculated. The passing frequency was determined from the formula [1 (molecule)] / [average of the time during which the nanopore was open (ms)]. When the probe concentration was 500 nM, the passing frequency was 1.37×10 -2 s -1 whereas when the probe concentration was 500 pM, the passing frequency was 2.96×10 -4 s -1 That is, it was confirmed that the higher the probe concentration, the higher the frequency of the probe passing through the nanopore, and it becomes easier to detect miRNA at extremely low concentrations.

[0211] From the above, in the examples, it was possible to determine the presence or absence of a specific oligonucleotide at an extremely low concentration (in the range of 1.0 aM to 1.0 pM) in the test sample.

Explanation of symbols

[0212] 1N nanopore Pore of 1P nanopore 2 lipid bilayer 3 first solution 4 second solution 5A, 5B, 5C, 5D, 5E specific oligonucleotide 6 probe 7 voltage application means

Claims

1. (1) A test sample containing a probe having a sequence complementary to one or more specific oligonucleotides and at least one of the specific oligonucleotides at a concentration of 1.0 aM to 1.0 pM is prepared by subjecting the test sample to a concentration ratio of the probe to the at least one oligonucleotide of 1.0 x 10 ４ : 1 to 1.0 x 10 ９ A first step of preparing a mixture of the above components so that the mixture is 1: (2) A second step of applying a voltage between the test sample as a first solution and a second solution, which are separated from each other by a lipid bilayer membrane having a nanopore, and measuring the current intensity of the current flowing between the first solution and the second solution over time to obtain current time-dependent change data; (3) a third step of determining the presence or absence of the at least one type of oligonucleotide in the test sample based on the current time course data; 1. A method for determining the presence or absence of at least one oligonucleotide at a concentration of 1.0 aM to 1.0 pM in a test sample, comprising:

2. The method of claim 1 , further comprising determining the amount of said at least one oligonucleotide.

3. The method of claim 1 or claim 2, wherein the one or more specific oligonucleotides comprise two or more specific oligonucleotides.

4. The method according to claim 3 , wherein the probe contains sequences complementary to each of the two or more specific oligonucleotides in one molecule.

5. the order of the sequences complementary to the two or more specific oligonucleotides in the probe is selected so as to minimize the free energy of the complex between the probe and the two or more specific oligonucleotides; The method according to claim 3 or claim 4.

6. The method of claim 3 , wherein the probes comprise two or more probes, each complementary to a different specific oligonucleotide.

7. Between the first step and the second step, Annealing the test sample; The method according to any one of claims 1 to 6.

8. The specific oligonucleotide is a miRNA, The probe is an oligodeoxyribonucleotide. The method according to any one of claims 1 to 7.

9. The probe comprises: ｘ at the 3' end, n is any deoxyribonucleotide, x is an integer from 3 to 30, and x n's are the same as each other. The method according to any one of claims 1 to 8.

10. The (n) ｘ The structure is a polydeoxycytosine structure.

10. The method of claim 9.

11. The probe has a hairpin structure at the 5' end. The method according to any one of claims 1 to 10.

12. (1-1) A probe having a sequence complementary to one or more specific oligonucleotides that are differentially expressed in a patient having a specific disease or an individual at risk of having a specific disease, or in the tissues of such individuals, and a sample derived from a test subject containing at least one of the specific oligonucleotides are subjected to a test in which the concentration of the at least one oligonucleotide in the test sample is 1.0 aM to 1.0 pM, and the ratio of the concentration of the probe to the concentration of the at least one oligonucleotide is 1.0 x 10 ４ : 1 to 1.0 x 10 ９ : Mix so that the ratio becomes 1 to obtain a test sample. Or, (1-2) A probe having a sequence complementary to one or more specific oligonucleotides that are not differentially expressed in a patient having a specific disease or an individual at risk of having a specific disease, or in the tissues thereof, and a sample derived from a test subject containing at least one of the specific oligonucleotides are subjected to a test in which the concentration of the at least one oligonucleotide in the test sample is 1.0 aM to 1.0 pM, and the ratio of the concentration of the probe to the concentration of the at least one oligonucleotide is 1.0 x 10 ４ : 1 to 1.0 x 10 ９ : Mix so that the ratio becomes 1 to obtain a test sample. The first step, (2) A second step of applying a voltage between the test sample as a first solution and a second solution, which are separated from each other by a lipid bilayer membrane having a nanopore, and measuring the current intensity of the current flowing between the first solution and the second solution over time to obtain current time-dependent change data; (3) a third step (excluding diagnostic procedures by a physician) of detecting the presence of the at least one type of oligonucleotide in the test sample based on the data on the change in current over time, thereby determining whether the test subject is suffering from a specific disease or is at risk of suffering from a specific disease; A method for determining whether a test subject is suffering from a specific disease or is at risk of suffering from a specific disease, comprising:

13. The method of claim 12, wherein the one or more specific oligonucleotides comprises two or more specific oligonucleotides.

14. The method according to claim 12 or 13, wherein the specific disease is bile duct cancer.

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