Method for determining accuracy of bisulfite treatment

Abstract

The present invention addresses the problem of providing a means for determining the quality of a bisulfite treatment reaction.　Provided is a method for evaluating the detection accuracy of methylated cytosine in a detection region on the basis of the success or failure of a bisulfite treatment performed on the detection region and a control region which contains unmethylated cytosine. This method allows for the determination of the quality of bisulfite treatment when assessing the presence or absence of methylation in cytosine being detected.

Description

Method for determining the accuracy of bisulfite processing

[0001] The present invention relates to a kit, device, and method for determining the accuracy of bisulfite (BS) treatment, and to a method for detecting methylation.

[0002] Most colorectal cancers are sporadic colorectal cancers that develop later in life due to exposure to environmental factors and the accumulation of gene mutations in the mucosal cells of the colon. For this reason, the incidence of sporadic colorectal cancer increases with age (Non-Patent Literature 1).

[0003] On the other hand, some types of colorectal cancer have a familial accumulation and are collectively referred to as hereditary colorectal cancer. Many of the mutations that cause hereditary colorectal cancer are found in tumor suppressor genes and mismatch repair genes. Generally, hereditary colorectal cancer is characterized by its onset at a young age and a high probability of developing multiple colorectal cancers and multi-organ cancers such as endometrial cancer (Non-patent Literature 1). Therefore, reliably identifying hereditary tumors is important for cancer prevention and early detection in that family.

[0004] Hereditary colorectal cancers include familial adenomatous polyposis (FAP) and Lynch syndrome-related tumors (hereditary non-polyposis colorectal cancer: HNPCC), which are frequently inherited in an autosomal dominant manner (Non-patent Literature 1).

[0005] FAP (Follow-onset Adenoma) is easily diagnosed based on its clinical characteristic of developing more than 100 adenomas in the colonic mucosa. On the other hand, Lynch syndrome-related tumors are thought to be likely to be overlooked in routine clinical practice because they do not show any clinical differences from sporadic colorectal cancer. However, Lynch syndrome-related tumors and sporadic colorectal cancer differ significantly in treatment strategies (surgery, chemotherapy, etc.) and prognostic examination methods (Non-patent Literature 1). Therefore, accurately diagnosing both in the early stages of clinical practice is extremely important.

[0006] Non-patent document 2 reported that methylation of CpG islands in the promoter region of the MLH1 gene encoding the MLH1 protein is the cause of MLH1 protein deficiency in sporadic colorectal cancer. On the other hand, methylation in the MLH1 protein promoter is not observed in Lynch syndrome-related tumors. Therefore, it is suggested that by using the blood of a subject and confirming the presence or absence of CpG island methylation in the MLH1 gene promoter as a biomarker, it may be possible to accurately determine whether the subject has sporadic colorectal cancer or Lynch syndrome-related tumors.

[0007] In addition, changes in DNA methylation status have been reported to be involved in many diseases, increasing the importance of accurately identifying DNA methylation status in disease testing, prevention, and treatment.

[0008] Guidelines for the Treatment of Hereditary Colorectal Cancer, 2024 Edition, Kinbara Publishing Co., Ltd. JG Herman, et al., 1998, Sci. USA, 95: 6870-6875.

[0009] Generally, the presence or absence of DNA methylation is detected by treating the target cytosine with bisulfite, converting unmethylated cytosine to uracil, and then detecting the difference in bases between the unmethylated and methylated cytosine.

[0010] However, if the bisulfite treatment is unsuccessful, the uracil conversion of unmethylated cytosine does not occur, and it remains as cytosine, leading to the misidentification of the target cytosine as methylated cytosine. This poses serious problems, such as leading to misdiagnosis in methylation-based diagnoses of sporadic colorectal cancer and Lynch syndrome-related tumors.

[0011] The object of this invention is to provide a means for determining the success or failure of a bisulfite treatment reaction.

[0012] To solve the above problems, the inventors have developed a method for evaluating the detection accuracy of methylated cytosine in the detection region based on the success or failure of bisulfite treatment of a control region containing unmethylated cytosine along with the detection region. This method makes it possible to determine the quality of the bisulfite treatment used for methylation detection when determining the presence or absence of methylation in the cytosine to be detected, thereby solving the above problems. The present invention is based on this newly developed method and includes the following.

[0013] [1] A kit for determining the accuracy of bisulfite (BS) treatment, comprising a non-BS treatment detection probe capable of binding to a control region containing one or more unmethylated cytosines in a control nucleic acid before BS treatment, and a BS treatment detection probe capable of binding to the same region in a control nucleic acid after BS treatment. [2] The kit according to [1], wherein the control region is a region that does not contain methylated cytosine (mC). [3] The kit according to [1] or [2], further comprising a control primer set for amplifying the control region in a control nucleic acid after BS treatment. [4] The kit according to [3], wherein the control primer set is capable of binding to a region that does not contain cytosine. [5] The kit according to any one of [1] to [4], further comprising a control nucleic acid. [6] A device for determining the accuracy of BS treatment, comprising a non-BS treatment detection probe capable of binding to a control region containing cytosine in a control nucleic acid before BS treatment, and a BS treatment detection probe capable of binding to the same region in a control nucleic acid after BS treatment. [7] A method for determining the accuracy of a BS treatment, comprising: a control BS treatment step of performing a BS treatment on a control nucleic acid; a control probe binding step of hybridizing a control probe, which is capable of binding to the control region and comprises a BS treatment detection probe and a non-BS treatment detection probe, to the control region after the control BS treatment; a control probe measurement step of measuring the amount of each probe bound after the control probe binding step; and a BS treatment determination step of determining whether the BS treatment is good or bad based on the binding ratio of the BS treatment detection probe and the non-BS treatment detection probe, wherein the control region contains cytosine and the number of mC is known. [8] The method according to [7], further comprising a control region amplification step of amplifying the control region using the control nucleic acid after the control BS treatment as a template by a nucleic acid amplification method using a control primer set capable of amplifying the control region, before the control probe binding step. [9] The method according to [7] or [8], wherein the determination is made by determining that the accuracy of the BS treatment was high if the binding ratio calculated using the following formula I is lower than a predetermined cutoff value.Binding ratio = Non-BS treated detection probe binding amount / (Non-BS treated detection probe binding amount + BS treated detection probe binding amount) Equation I

[10] A method for detecting methylation in the detection region of a test nucleic acid, comprising: a BS treatment step of BS treating a test nucleic acid and a control nucleic acid; a control probe binding step of hybridizing a control probe, which consists of a BS treated detection probe and a non-BS treated detection probe, to the control region after the BS treatment, which is capable of binding to the control region of the control nucleic acid; a control probe measurement step of measuring the binding amount of each probe after the control probe binding step; a BS treatment determination step of determining the quality of the BS treatment based on the binding amount of the BS treated detection probe and the binding amount ratio of the non-BS treated detection probe; and using the test nucleic acid after the BS treatment step as a template, the detection region, its detection region A method comprising: a detection region amplification step of amplifying the region using a set of test primers capable of amplifying the region; a test probe binding step of hybridizing an mC-compatible probe and a non-mC-compatible probe to the amplified product after the detection region amplification step; a test amount measurement step of measuring the amount of each probe bound after the test probe binding step; and a methylation determination step of determining whether or not methylation is present in the detection region based on the binding ratio of the mC-compatible probe and the non-mC-compatible probe, wherein the control region contains cytosine, the number of mCs is known, and the methylation determination step is performed only if the BS treatment is determined to be good in the BS treatment step. This specification encompasses the disclosures of Japanese Patent Application No. 2025-008760, which forms the basis of the priority of this application.

[0014] According to the kit, device, and method for determining the accuracy of bisulfite treatment of the present invention, the quality of bisulfite treatment can be easily determined.

[0015] According to the methylation detection method of the present invention, when determining whether or not methylation is present in the detection region of the test nucleic acid, it is possible to determine the quality of the bisulfite treatment used for methylation detection.

[0016] This figure shows the principle and problems of conventional bisulfite (BS) treatment for detecting DNA methylation. (a) shows the changes in the base sequence of methylated nucleic acids, and (b) shows the changes in the base sequence of unmethylated nucleic acids. In the figure, m "C" represents methylated cytosine (mC), and the square frame indicates the location of an exemplary methylation target site. This figure schematically shows how to determine the success or failure of bisulfite (BS) treatment using the control probe of the present invention. (a) shows whether each probe can bind when the BS reaction is successful, and (b) shows whether each probe can bind when the BS reaction is unsuccessful. In the figure, "+" indicates that the probe can bind, and "-" indicates that the probe cannot bind. m "C" indicates methylated cytosine (mC), and the square frame indicates the location of an exemplary control region. This figure schematically shows how to determine the quality of bisulfite (BS) treatment using the test probe of the present invention. (a) shows whether each probe can bind when the detection region contains methylated cytosine at the methylation target site, and (b) shows whether each probe can bind when the detection region does not contain methylated cytosine at the methylation target site. In the figure, "+" indicates that the probe can bind, and "-" indicates that the probe cannot bind. m "C" indicates methylated cytosine (mC), and the square frame indicates the location of an exemplary detection region. This is an exemplary flowchart of the accuracy determination method of the present invention. This is an exemplary flowchart of the methylation detection method of the present invention. This is a flowchart of the control region performed within the quantitative nucleic acid amplification step (S0203) in the flowchart shown in Figure 5. This is a flowchart of the detection region performed within the quantitative nucleic acid amplification step (S0203) in the flowchart shown in Figure 5. This is a plot diagram showing the relationship between the methylation rate and the judgment value in the MLH1 gene promoter. A shows the result for the promoter C region, and B shows the result for the promoter D region. In the figure, ○ indicates genomic DNA derived from peripheral blood, and ● indicates plasmid DNA used as control nucleic acid. The judgment value (0.1) shown by the dashed line in the figure indicates the cutoff value that separates methylation rates of 0% and 5%.

[0017] 1. Kit and Device for Determining the Quality of Bisulfite (BS) Treatment 1-1. Overview A first aspect of the present invention is a kit for determining the quality of bisulfite (BS) treatment (often abbreviated as "quality determination kit" herein). The quality determination kit of the present invention includes a BS treatment detection probe and a non-BS treatment detection probe as essential components, and also includes a control primer set, control nucleic acid, and detection reagent as selective components. By using the quality determination kit of the present invention, the quality of bisulfite treatment can be determined.

[0018] 1-2. Definitions The following terms used herein are defined: "Bisulfite treatment" (often referred to as "BS treatment" herein) is a method of reacting DNA with bisulfite to convert unmethylated cytosine (n-mC) on the DNA to uracil (U). As shown in Figure 1, this base conversion changes the base at that position to thymine (T) after the amplification reaction. On the other hand, methylated cytosine (mC) is not affected by bisulfite and no base conversion occurs, so its position remains cytosine even after the nucleic acid amplification reaction following the treatment. In this way, by comparing the base sequences of DNA with and without bisulfite treatment and nucleic acid amplification, the presence or absence of methylation of cytosine on the DNA can be easily identified.

[0019] In this specification, "methylation" refers to the methylation of DNA. More specifically, it refers to the methylation of cytosine in the CpG sequence on DNA.

[0020] In this specification, "methylated cytosine" (often denoted as "mC" herein) refers to cytosine in a CpG sequence that has been methylated, or the methylated cytosine itself. Specifically, it refers to cytosine in which the carbon at position 5 of the cytosine in the CpG sequence has been methylated by DNA methyltransferase (DNMT) and converted to 5-methylcytosine (5mC).

[0021] In this specification, "non-methylated cytosine" (often denoted as "n-mC") refers to cytosine that is not actually methylated. In principle, this includes cytosines other than those in CpG sequences and cytosines in CpG sequences that are not methylated. However, even cytosines that can be methylated, such as those in CpG sequences, may be referred to as n-mC if, for example, they are actually methylated but not converted to U due to a poor reaction during bisulfite treatment.

[0022] A "CpG sequence" refers to a dinucleotide sequence consisting of a phosphodiester bond between cytosine and guanine. As mentioned above, cytosine in a CpG sequence is the target of methylation.

[0023] A "CpG island" refers to a region on DNA where CpG sequences appear frequently. Assuming there is no bias in the types of bases (A, G, C, and T) appearing in a given genomic region, the probability of a CpG sequence appearing is 1 / 16. Therefore, a CpG island can be defined as a region in a given genomic region where the probability of a CpG sequence appearing exceeds 1 / 16. However, this specification is not limited to this definition, and a CpG island may also be a region in which the GC content in the given genomic region exceeds 50% and the proportion of CpG sequences present is 60% or more of the amount expected from the GC content (CpG observed / expected ≥ 0.6).

[0024] In this specification, "control nucleic acid" refers to a nucleic acid containing a control region used to confirm the effectiveness of the BS treatment. This nucleic acid is, in principle, DNA.

[0025] In this specification, "control region" refers to a base sequence region contained in the control nucleic acid that is subject to detection for confirming the effectiveness of the BS treatment reaction. Specifically, it refers to a base sequence containing n-mC, and in particular, a base sequence region containing one or more cytosines other than CpG that are not likely to be methylated.

[0026] In this specification, "test nucleic acid" refers to the nucleic acid contained in the sample to be tested for the presence or absence of methylation.

[0027] In this specification, "detection region" refers to the base sequence region in the test nucleic acid that is subject to detection for the presence or absence of methylation.

[0028] In this specification, "methylation target site" refers to the cytosine of the CpG sequence that is the target of methylation.

[0029] In this specification, "simultaneous determination" does not mean making determinations at the same time, i.e., at the same moment, but rather means determining the presence or absence of methylation, and at the same time determining the quality of the bisulfite treatment used to detect that methylation.

[0030] 1-3. Structure The methylation detection kit of the present invention includes a BS-treated detection probe and an n-BS-treated detection probe as essential components, which are control probes. Each component will be described in detail below.

[0031] (1) Bisulfite-treated detection probe (BS-treated detection probe) The "bisulfite (BS)-treated detection probe" is one of the control probes and hybridizes to a base sequence containing one or more bases corresponding to n-mCs in the control region after BS treatment (Figure 2(a)). "Bases corresponding to n-mCs" refers to the bases corresponding to the n-mCs in the nucleic acid before or after BS treatment. Since all n-mCs in the control region are converted to uracil by BS treatment, in the control nucleic acid containing the control region after BS treatment or in nucleic acid amplification products using it as a template, the bases corresponding to the n-mCs become thymine. The BS-treated detection probe hybridizes to a base sequence in the control region after BS treatment that contains a site where n-mCs have been converted, for example, a site where cytosine has been converted to thymine.

[0032] The base sequence of the BS treatment detection probe includes, for example, a base sequence complementary to the following base sequences: (i) a base sequence containing a control region, wherein the base corresponding to n-mC in the control region is thymine; (ii) a base sequence in which one or more bases other than the base corresponding to n-mC in the control region in the base sequence of (i) are added, deleted, and / or substituted; (iii) a base sequence having a sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more with the base sequence of (i), and the base corresponding to n-mC in the control region is thymine; or (iv) a base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence of (i).

[0033] In addition, when the control region contains mC, the base sequence of the BS treatment detection probe may further include a base sequence complementary to a base sequence in which the base corresponding to mC is cytosine. Specific examples of the base sequence in this case include, for example, a base sequence in which the base corresponding to mC in the control region is cytosine in the base sequences of (i) to (iv).

[0034] Also, as the base sequence of the BS treatment detection probe, for example, a base sequence that does not hybridize with a base sequence in which the base corresponding to n-mC in the control region is cytosine under stringent conditions can be preferably used.

[0035] As used herein, the term "a plurality" refers to 2 to 10, for example, 2 to 7, 2 to 5, 2 to 4, 2 to 3 (several).

[0036] As used herein, "nucleotide (base) sequence identity" refers to a numerical value indicating the ratio of sites where the types of nucleotides are identical within the comparison range of two nucleotide sequences. Nucleotide sequence identity can be calculated by aligning the two nucleotide sequences so that the nucleotide match degree within the comparison range is maximized, even when the lengths of the two nucleotide sequences are different. Without limitation, a typical algorithm for performing such analysis is BLAST. BLAST is available in various software and web services. For example, nucleotide sequence identity can be easily calculated using genetic information processing software GENETYX (https: / / www.genetyx.co.jp / ), the BLAST server provided by NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), etc. In addition to BLAST, there are also algorithms such as FASTA, etc., which can be used as long as appropriate identity can be calculated, and the method used is not particularly limited.

[0037] "Stringent conditions" refer to conditions under which non-specific hybridization does not occur or occurs rarely. Specifically, for example, after performing a hybridization reaction at 50°C for 16 hours, washing is carried out under the conditions of 25°C for 10 minutes and 2×SSC, 25°C for 5 minutes in 2×SSC / 0.2% SDS, 5×Denhardt's reagent, and 100 μg / mL denatured and fragmented DNA (e.g., salmon sperm DNA: ssDNA). It may also be carried out under more highly stringent conditions. "Highly stringent conditions" refer to conditions for a more rigorous hybridization reaction, which means performing hybridization and washing under conditions of low salt concentration and / or high temperature. Specifically, for example, incubating with a probe at 65°C to 68°C in 6×SSC / 0.5% SDS, 5×Denhardt's reagent, and 100 μg / mL ssDNA, and then starting from room temperature in a washing solution of 2×SSC, 0.1% SDS, lowering the salt concentration in the washing solution to 0.1×SSC, and raising the temperature to 68°C until no background signal is detected.

[0038] BS-treated detection probes are composed of native nucleotides and / or non-native nucleotides. Typically, they consist of native nucleotides consisting of DNA only, RNA only, or a hybrid of DNA and RNA, but DNA only is preferred due to its chemical stability and ease of chemical synthesis. Furthermore, if necessary, native nucleotides of DNA and RNA can be combined, or non-native nucleotides such as chemically modified nucleic acids or pseudo-nucleic acids can be added to some of them. Examples of chemically modified nucleic acids and pseudo-nucleic acids include PNA (Peptide Nucleic Acid), BNA (Bridged Nucleic Acid) / LNA (Locked Nucleic Acid; registered trademark), methylphosphonate-type DNA, phosphorothioate-type DNA, and 2'-O-methyl-type RNA.

[0039] The BS-treated detection probe only needs to be designed to specifically hybridize with cytosine (n-mC) sequences that are not methylation target sites in the control region at the time of probe application, and is not particularly limited. Typically, it is sufficient if the probe is designed to be complementary to the nucleotide sequence of the control region at the time of probe application. Alternatively, sequences other than n-mC may be hybridizable with the nucleotide sequence of the control region under stringent conditions.

[0040] The Tm value of the BS processing detection probe is not particularly limited, but it is preferably in the range of 55°C to 80°C or 60°C to 75°C.

[0041] The base length of the BS-treated detection probe should be such that it can hybridize to a base sequence containing all or part of n-mC in the control region after BS treatment. For example, the probe base length can be one of the following, commonly used in this field: 15 to 100 consecutive bases, 15 to 80 consecutive bases, 15 to 60 consecutive bases, 16 to 50 consecutive bases, 17 to 40 consecutive bases, 18 to 30 consecutive bases, or 20 to 25 consecutive bases.

[0042] BS-treated detection probes may have some or all of the phosphate groups, sugars, and / or bases of the nucleotides that constitute them labeled with a labeling substance. The labeling position of the labeling substance on the probe can be appropriately determined according to the characteristics of the labeling substance and the intended use, and is not limited, but the 5' end, 3' end, and / or the SNP site to be detected are preferred. Any substance known in the art can be used as the labeling substance. Examples include DIG, fluorescent substances, quenchers, chemiluminescent substances, radioisotopes, biotin, or magnetic beads. Labeling of nucleotides with each labeling substance can be carried out by known methods. The types of labeling on other primers and probes are basically the same as those on the BS-treated detection probe and are not particularly limited. These labels may be used on primers or probes as needed.

[0043] The term "fluorescent substance" refers to a substance that becomes excited by absorbing excitation light of a specific wavelength and emits fluorescence when returning to its original ground state. In this specification, fluorescent substances are included. Examples include FITC, Texas, Cy3, IC3, Cy5, IC5, Cy7, IC7, FAM, HEX, VIC, JOE, ROX, TET, Bodipy493, NBD, TAMRA, Quasar® 670, Quasar® 705, CAL Fluor® Red 610, fluorescein or its derivatives, or rhodamine or its derivatives.

[0044] The term "quencher" refers to a substance that absorbs the excitation energy of the fluorescent substance and suppresses its fluorescence. Examples include AMRA, DABCYL, BHQ-1, BHQ-2, or BHQ-3. Generally, quenchers are used in combination with fluorescent substances. Since different types of quenchers have different fluorescence suppression wavelength ranges, when combining a fluorescent substance with a quencher, it is important to use a quencher that can suppress the fluorescence of the fluorescent substance used for labeling. For example, if the fluorescent substance is FAM, HEX, TET, etc., it should be combined with BHQ1, which has a suppression wavelength range of 480 nm to 580 nm. If the fluorescent substance is Cy3, IC3, Cy5, IC5, ROX, TAMRA, Texas, etc., it should be combined with BHQ2, which has a suppression wavelength range of 550 nm to 650 nm. The arrangement of the fluorescent substance and quencher in the probe is not particularly limited, as long as both substances are arranged so that the quencher can suppress the fluorescence produced by the fluorescent substance. For example, one end of the probe (either the 5' or 3' end) can be labeled with a fluorescent substance, and the other end (either the 3' or 5' end) can be labeled with a quencher.

[0045] The aforementioned "chemiluminescent material" refers to a substance that, after being excited by a chemical reaction, releases the difference in energy as light when returning to its ground state. Examples include luminol, rofin, and lucigenin.

[0046] The aforementioned "radioactive isotopes" refer to isotopes with different mass numbers that emit radiation. For example, 32 P, 33 P, or 35 S is one example.

[0047] As a specific example of the BS-treated detection probe, if the control nucleic acid is the β-actin gene, a polynucleotide consisting of the base sequence shown in Sequence ID No. 10 can be used, which hybridizes to the amplification product obtained after nucleic acid amplification using the control primer set (n-mC-Fw / n-mC-Rv) described later, using the β-actin gene after BS treatment as a template.

[0048] (2) Non-bisulfite-treated detection probe (n-BS-treated detection probe) The "non-bisulfite (n-BS)-treated detection probe" is one of the control probes, and is a probe that pairs with the BS-treated detection probe, and hybridizes to a base sequence containing a base corresponding to n-mC in the control region that has not been treated with bisulfite (Figure 2(b)).

[0049] If BS treatment is not performed, the n-mCs in the control region will not be converted to uracil. Therefore, in control nucleic acids containing the control region or nucleic acid amplification products using the control region as a template, all bases corresponding to the n-mCs remain cytosine.

[0050] The n-BS-treated detection probe is configured to hybridize to a base sequence containing the same n-mC corresponding to the same control region in the same control nucleic acid, for use in pair with the BS-treated detection probe. However, the base sequences and base lengths other than the specific cytosine to be detected may be the same or different in both probes. Furthermore, when two or more sets of BS-treated detection probes and n-BS-treated detection probes are included, each set is, in principle, configured to hybridize to a base sequence containing the same n-mC corresponding to the same base, or to a base sequence containing the same n-mC corresponding to the same base.

[0051] The nucleotide sequences of the n-BS-treated detection probes include, for example, nucleotide sequences complementary to the following nucleotide sequences: (i') A nucleotide sequence that includes a control region, wherein the base corresponding to n-mC in the control region is cytosine; (ii') A nucleotide sequence in which one or more bases other than the base corresponding to n-mC in the control region are added, deleted, and / or substituted in the nucleotide sequence of (i'); (iii') A nucleotide sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more sequence identity with the nucleotide sequence of (i'), wherein the base corresponding to n-mC in the control region is cytosine; or (iv') A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of (i').

[0052] Furthermore, if the control region includes mC, the base sequence of the n-BS-treated detection probe may also include a base sequence complementary to the base sequence in which the base corresponding to the mC is cytosine. Specific examples of such base sequences include, for example, the base sequences (i') to (iv') in which the base corresponding to the mC in the control region is cytosine.

[0053] Furthermore, as the base sequence of the n-BS-treated detection probe, for example, a base sequence that does not hybridize under stringent conditions with a base sequence in which the base corresponding to n-mC in the control region is thymine can be suitably used.

[0054] A pair of BS-treated detection probes and n-BS-treated detection probes hybridize to base sequences containing the same n-mC-corresponding bases in the control region. However, the base sequences of the nucleic acid amplification product of the control region after BS treatment differ from those of the nucleic acid amplification product of the control region that has not been treated with BS (i.e., not subjected to or affected by BS treatment). Therefore, the BS-treated detection probe can only hybridize to the nucleic acid amplification product of the control region after BS treatment (Figure 2(a)), and the n-BS-treated detection probe can only hybridize to the nucleic acid amplification product of the control region that has not been treated with BS (Figure 2(b)). By utilizing this difference, when the nucleic acid amplification product of the control nucleic acid after BS treatment is mixed with each probe, it is possible to determine whether the BS treatment was successful or unsuccessful based on which probe was able to hybridize (Figure 2).

[0055] The basic design and configuration (including labeling with a labeling substance) of the n-BS processed detection probe are the same as those of the BS processed detection probe described above, so a detailed explanation is omitted here.

[0056] As a specific example of the n-BS treatment detection probe, if the control nucleic acid is a β-actin gene, a polynucleotide consisting of the base sequence shown in Sequence ID No. 11 can be used to hybridize the amplification product obtained after nucleic acid amplification using the aforementioned control primer set (n-mC-Fw / n-mC-Rv) with an untreated β-actin gene as a template. By using the n-BS treatment detection probe shown in Sequence ID No. 11 in pair with the BS treatment detection probe shown in Sequence ID No. 10, it is possible to determine the quality of the BS treatment in the control nucleic acid after BS treatment.

[0057] (3) Control primer set A "control primer set" consists of one or more primers configured to hybridize to the base sequence of a control nucleic acid and amplify the control region.

[0058] The control region is included in the control nucleic acid and is the target for detection to confirm the success or failure of the BS treatment reaction. It includes base sequences containing n-mC, particularly base sequence regions containing one or more cytosines other than CpG that are unlikely to be methylated.

[0059] A control primer set can be appropriately designed according to common practices in the field, such that the base sequence of the control region containing n-mC is located within the base sequence region (excluding the primer sequence) that is amplified by the primer set. The primers are preferably designed to hybridize to sequences unaffected by BS treatment, such as sequences without n-mC or sequences without C. While not limited to these, it is preferable that the region to which the primers hybridize in the design of the control primer set does not contain unmethylated cytosine. If the region to which the primers hybridize contains unmethylated cytosine, it is converted to uracil by BS treatment and then to thymine by subsequent quantitative nucleic acid amplification. Therefore, if the control primer set is to hybridize to both strands, two sets are required: one for BS treatment and one for n-BS treatment. However, if the region to which the primers hybridize does not contain unmethylated cytosine, one set of primers is sufficient, regardless of whether BS treatment or n-BS treatment is performed.

[0060] The amplified control region is as detailed in the description of the control nucleic acid. The amplified sequence may include any sequence in addition to the control region.

[0061] The base length of the amplification product is not particularly limited as long as it is long enough to amplify the detection region. For example, it can be 40-1000 bases, 40-800 bases, 50-500 bases, 50-300 bases, 60-270 bases, 60-200 bases, 70-150 bases, 70-100 bases, etc.

[0062] Each primer can be appropriately designed according to the common practices of the art so that the target n-mC is located within the nucleotide sequence region (excluding the primer sequence) that is amplified by the primer set. Forward (Fw) primers can be designed, for example, 5' to the 5' end of the control region, at a distance of, for example, 1 nucleotide or more, 3 nucleotides or more, 5 nucleotides or more, 10 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, or 30 nucleotides or more. Similarly, reverse (Rv) primers can be designed 3' to the 3' end of the control region, and on the complementary (antisense) strand, at a distance of, for example, 1 nucleotide or more, 3 nucleotides or more, 5 nucleotides or more, 10 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, or 30 nucleotides or more.

[0063] The Fw primer can be a polynucleotide consisting of 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more consecutive base sequences in the control region, or a polynucleotide containing one or more deletions, substitutions, or additions of bases in the base sequence of the said polynucleotide, or a polynucleotide containing 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more consecutive base sequences in the control region. This Fw primer can hybridize to the base sequence on the complementary side of the control region on the control nucleic acid.

[0064] The Rv primer can be a polynucleotide consisting of 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more consecutive base sequences on the complementary side of the control region, or a polynucleotide containing one or more deletions, substitutions, or additions of bases in the base sequence of the said polynucleotide, or a polynucleotide containing 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more consecutive base sequences on the complementary side of the control region. This Rv primer can be hybridized to the base sequence of the control region on the control nucleic acid.

[0065] It is preferable that the Fw primer and Rv primer be designed so that their respective Tm values ​​are in the range of 55°C to 80°C or 60°C to 75°C.

[0066] Each primer may include an additional sequence at its 5' end that differs from the base sequence of the template nucleic acid. The additional sequence can be any base sequence, as long as it does not self-anneal and does not undergo hybridization or other interactions with the target nucleic acid or other primers or probes included in the kit of the present invention. Examples include base sequences containing restriction enzyme sites or new primer binding sites.

[0067] Each primer consists of native and / or non-native nucleotides. Their details are described in the description for the BS-treated detection probe.

[0068] Furthermore, each primer may have some or all of its constituent nucleotides—phosphate groups, sugars, and / or bases—labeled with a labeling substance. Details regarding the labeling substance are the same as those described for the BS-treated detection probe. Note that in the control primer set used in the examples described later, the R primer (ACT-R) is IC5-labeled.

[0069] The labeling position of the labeling substance in the primer can be determined appropriately according to the properties of the labeling substance and its intended use, and is not limited, but the 5' end, which does not contribute to the extension reaction, is usually preferred.

[0070] Specific examples of control primer sets include, for instance, the Fw primer (ACT-F) shown in SEQ ID NO: 8 and the Rv primer (ACT-R) shown in SEQ ID NO: 9, when the control nucleic acid is the β-actin gene.

[0071] (4) Control nucleic acid The control nucleic acid is a nucleic acid that contains a control region to confirm the quality of the BS treatment reaction, as described above. This control nucleic acid only needs to contain a control region containing one or more unmethylated cytosines.

[0072] The type of control nucleic acid is not limited. For example, it may be an artificial nucleic acid or a natural nucleic acid. In the case of an artificial nucleic acid, its composition is not particularly limited. For example, the BS-treated detection probe may contain the natural and / or non-natural nucleotides mentioned above, and may be labeled with a labeling substance. Furthermore, its sequence may be a natural sequence, a sequence mimicking it, or an artificially designed sequence. For example, the amplification product of a natural sequence does not contain methylated cytosine and can therefore be used as a control nucleic acid.

[0073] Natural nucleic acids may be, for example, nucleic acids in a sample containing the test nucleic acid, and specifically include, for example, genomic DNA, mitochondrial DNA, chloroplast DNA, or fragments thereof (cfDNA, etc.). When the control nucleic acid is natural DNA, the control region may be, for example, another gene or its regulatory region in that natural DNA that is different from the gene to which the detection region belongs, or a nucleic acid region other than a gene or regulatory region (e.g., a spacer region), or a region other than the detection region or the same region as the detection region in the gene to which the detection region belongs. However, if the control region is the same as the detection region, the control nucleic acid is a different nucleic acid from the test nucleic acid, and the number and position of unmethylated cytosines in the control region are known.

[0074] The control nucleic acid may be the same molecule as the test nucleic acid, a different molecule, or a different type of molecule (DNA). When the control nucleic acid is the same molecule as the test nucleic acid, it means that a specific nucleic acid region within a given nucleic acid molecule is used as the control region, and another nucleic acid region within the same molecule is used as the detection region. In this case, this nucleic acid molecule is referred to as both the control nucleic acid and the test nucleic acid.

[0075] The number of unmethylated cytosines in the control region is not particularly limited, as long as it is one or more, but its number and location are known. The sequence of the control region is not particularly limited, but may include, for example, a methylated cytosine (mC)-free region, a region without CpG islands, a region without CpG sequences, or a structural gene region. If the control gene is a structural gene region, the specific gene is not particularly limited, but examples include housekeeping genes such as the β-actin (ACTB) gene.

[0076] There may be one control region or multiple control regions. When using multiple control regions, all control regions may be contained in the same control nucleic acid, or one or more control regions may be contained in a different control nucleic acid than the others. Furthermore, all control regions may be of the same type, or one or more control regions may be of a different type than the others.

[0077] If included in this kit, the control nucleic acid is an exogenous polynucleotide added to the target sample.

[0078] (5) Detection reagents, etc. In addition to the above components, the accuracy determination kit of the present invention may also include, for example, reagents necessary for reverse transcription reactions and nucleic acid amplification reactions (RTase, dNTPs, Taq polymerase, etc.), reagents for detecting amplified nucleic acid fragments (fluorescent / luminescent reagents, etc.), and protocols describing methods for detecting appropriate reaction conditions, etc.

[0079] (6) The microarray accuracy determination kit of the present invention may be in the form of a device having a microarray (nucleic acid array, DNA chip) configuration.

[0080] For example, there is a configuration in which BS treatment detection probes and 5'-ends of n-BS treatment detection probes are arranged and immobilized at high density in a small spot shape on a carrier (chip) serving as a substrate.

[0081] As the material of the carrier, those known in the art can be used and are not particularly limited. For example, noble metals (including platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten, and their compounds, etc.), conductive materials such as carbon (including graphite, carbon fiber), silicon materials (including single crystal silicon, amorphous silicon, silicon carbide, silicon oxide, silicon nitride, and SOI), inorganic materials (including glass, quartz glass, alumina, sapphire, ceramics, forsterite, photosensitive glass, etc.), and organic materials (including polyethylene, ethylene, polypropylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene-acrylonitrile copolymer, acrylonitrile-butadiene-styrene copolymer, polyphenylene oxide, and polysulfone) and the like can be mentioned.

[0082] Although not limited, a carrier having a carbon layer and a chemical modification group on the surface is preferable. Such carriers include those having a carbon layer and a chemical modification group on the surface of the carrier, and those having a chemical modification group on the surface of a carrier composed of a carbon layer.

[0083] The form of the carrier is not particularly limited, but in a microarray, a flat plate structure is preferable. The shape of the carrier is not limited to rectangular, square, circular, etc., but usually 1 mm 2 to 75 mm 2 、2 mm 2 to 10 mm 2 、or 3 mm 2 to 5 mm 2Such materials are preferred. From the standpoint of being easy to manufacture into a flat plate structure, carriers made of silicon material or resin material are preferred, and carriers having a carbon layer and chemical modification groups on the surface of single-crystal silicon are particularly preferred.

[0084] The carbon layer formed on the support is not particularly limited, but it is preferable to use any of the following: synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (e.g., diamond-like carbon), amorphous carbon, carbon-based material (e.g., graphite, fullerene, carbon nanotube), a mixture thereof, or a laminate thereof. Carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, vanadium carbide, etc. The carbon layer is advantageous in that it has excellent chemical stability and can withstand subsequent chemical modification group introduction and reactions in bonding with the analyte, the bonding with the analyte is flexible because it is bonded by electrostatic coupling, it is transparent to UV detection system UV because it does not absorb UV, and it can conduct electricity during electroblotting.

[0085] Carbon layers can be formed by known methods. Examples include microwave plasma CVD (Chemical vapor deposit), ECRCVD (Electric cyclotron resonance chemical vapor deposit), ICP (Inductive coupled plasma), DC sputtering, ECR (Electric cyclotron resonance) sputtering, ionization deposition, arc deposition, laser deposition, EB (Electron beam) deposition, and resistance heating deposition.

[0086] In high-frequency plasma CVD, a glow discharge generated between electrodes by high frequency decomposes the raw material gas (methane), and a carbon layer is synthesized on the support. In ionization evaporation, thermionic electrons generated by a tungsten filament are used to decompose and ionize the raw material gas (benzene), and a carbon layer is formed on the support by a bias voltage. The carbon layer may also be formed by ionization evaporation in a mixed gas consisting of 1 to 99 volume percent hydrogen gas and 99 to 1 volume percent of the remaining methane gas.

[0087] In arc evaporation, a DC voltage is applied between a graphite material (cathode evaporation source) and a vacuum vessel (anode) to induce an arc discharge in a vacuum, generating a plasma of carbon atoms from the cathode. By applying an even more negative bias voltage to the support than that applied to the evaporation source, carbon ions in the plasma are accelerated toward the support, thereby forming a carbon layer.

[0088] In laser deposition, for example, a carbon layer can be formed by irradiating a graphite target plate with pulsed Nd:YAG laser light to melt it and depositing carbon atoms onto a glass support.

[0089] When forming a carbon layer on the support surface, the thickness of the carbon layer is usually around a single molecular layer to 100 μm. If it is too thin, the surface of the underlying substrate may be locally exposed, and if it is too thick, productivity will be poor. Therefore, it is preferably 2 nm to 1 μm, and more preferably 5 nm to 500 nm.

[0090] By introducing chemically modifying groups to the surface of the support on which the carbon layer is formed, each probe can be firmly immobilized on the support. The chemically modifying groups to be introduced can be appropriately selected by those skilled in the art and are not particularly limited, but examples include amino groups, carboxyl groups, epoxy groups, formyl groups, hydroxyl groups, and active ester groups.

[0091] The introduction of amino groups can be carried out, for example, by irradiating the carbon layer with ultraviolet light in ammonia gas, or by plasma treatment. Alternatively, it can be carried out by chlorinating the carbon layer with ultraviolet light in chlorine gas, and then irradiating it with ultraviolet light again in ammonia gas. Alternatively, it can be carried out by reacting the chlorinated carbon layer with polyhydric amine gases such as methylenediamine and ethylenediamine.

[0092] The introduction of carboxyl groups can be carried out, for example, by reacting a suitable compound with the aminated carbon layer as described above. Examples of compounds used to introduce carboxyl groups include those with formula XR 1- COOH (where X is a halogen atom, R is a halogen atom) 1 Halocarboxylic acids represented by (where represents a divalent hydrocarbon group with 10 to 12 carbon atoms), such as chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid; formula: HOOC-R 2 -COOH (in the formula, R 2 Dicarboxylic acids represented by a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms, such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, and phthalic acid; polycarboxylic acids such as polyacrylic acid, polymethacrylic acid, trimellitic acid, and butanetetracarboxylic acid; formula: R 3 -CO-R 4 -COOH (in the formula, R 3 R is a hydrogen atom or a divalent hydrocarbon group having 1 to 12 carbon atoms. 4 Keto acids or aldehyde acids represented by (where represents a divalent hydrocarbon group having 1 to 12 carbon atoms); formula: X-OC-R 5 -COOH (where X is a halogen atom, R is a halogen atom) 5 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms. Examples include monohalides of dicarboxylic acids represented by ), such as succinic acid monolide and malonic acid monolide; and acid anhydrides such as phthalic anhydride, succinic anhydride, oxalic anhydride, maleic anhydride, and butanetetracarboxylic anhydride.

[0093] The introduction of epoxy groups can be carried out, for example, by reacting a suitable polyvalent epoxy compound with the aminated carbon layer as described above. Alternatively, it can be obtained by reacting an organic peracid with the carbon-carbon double bonds contained in the carbon layer. Examples of organic peracids include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, and trifluoroperacetic acid.

[0094] The introduction of the formyl group can be carried out, for example, by reacting glutaraldehyde with the aminated carbon layer as described above.

[0095] The introduction of hydroxyl groups can be carried out, for example, by reacting water with the chlorinated carbon layer as described above.

[0096] Active ester groups refer to esters that have a highly acidic electron-withdrawing group on the alcohol side of the ester group, thereby activating nucleophilic reactions; in other words, ester groups with high reactive activity. These are ester groups that have an electron-withdrawing group on the alcohol side of the ester group and are more active than alkyl esters. Active ester groups are reactive with groups such as amino groups, thiol groups, and hydroxyl groups. More specifically, phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, and esters of heterocyclic hydroxy compounds are known to be active ester groups that have much higher activity than alkyl esters, etc. More specifically, examples of active ester groups include p-nitrophenyl group, N-hydroxysuccinimide group, succinimide group, phthalimide group, and 5-norbornene-2,3-dicarboximide group, with N-hydroxysuccinimide group being particularly preferred.

[0097] The introduction of an active ester group can be carried out, for example, by activating the carboxyl group introduced as described above with a dehydration condensation agent such as cyanamide or carbodiimide (e.g., 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide) and a compound such as N-hydroxysuccinimide. This process makes it possible to form a group in which an active ester group such as an N-hydroxysuccinimide group is bonded to the terminal of the hydrocarbon group via an amide bond (Japanese Patent Application Publication No. 2001-139532).

[0098] In a microarray, a control nucleic acid treated with BS is reacted with each probe on the microarray. Detection and quantification can be achieved by detecting and measuring fluorescence based on hybridization of the target nucleic acid, etc., using a microplate reader or scanner. The quality of the BS treatment can be determined from the ratio of fluorescence intensities detected by the BS-treated detection probe and the n-BS-treated detection probe, and a preset threshold. If methylation is to be determined simultaneously, the dedicated software attached to the measuring device should be programmed to prompt remeasurement without determining the presence or absence of methylation if the treatment is inadequate.

[0099] (7) Test probe The kit or device of this embodiment further comprises a test probe and / or a test primer and can be a kit or device for methylation detection. With this kit or device, when determining whether or not methylation is present in the detection region of the test nucleic acid, it is possible to determine the quality of the bisulfite treatment used for methylation detection.

[0100] The "test probe" is a probe that hybridizes to a base sequence containing the methylation target site in the detection region (Figure 3).

[0101] The basic design and configuration of the test probe (including labeling with a labeling substance) are the same as those of the control probe, so a detailed explanation is omitted here.

[0102] The test probe is not particularly limited, as long as it is designed to specifically hybridize with the nucleotide sequence containing the methylation target site of the detection region at the time of probe application. Typically, it is sufficient if the probe is designed to have a nucleotide sequence complementary to the nucleotide sequence of the detection region at the time of probe application. Alternatively, the nucleotide sequence may have parts other than the methylation target site that can hybridize with the nucleotide sequence of the detection region under stringent conditions.

[0103] The Tm value of the test probe is preferably in the range of 55°C to 80°C, or 60°C to 75°C.

[0104] The base length of the test probe should be such that it can hybridize to all or part of the base sequence containing cytosine at a specific methylation target site in the amplified product of the detection region after BS treatment and nucleic acid amplification reaction. For example, the probe base length may be one of the following commonly used in this field: 15 to 100 consecutive bases, 15 to 80 consecutive bases, 15 to 60 consecutive bases, 16 to 50 consecutive bases, 17 to 40 consecutive bases, 18 to 30 consecutive bases, or 20 to 25 consecutive bases.

[0105] The test probe may contain other nucleotide sequences in addition to the nucleotide sequence complementary to the nucleotide sequence of the detection region after BS treatment. In this case, it is preferable that the other nucleotide sequences are located at the 5' end and / or 3' end of the nucleotide sequence complementary to the detection region.

[0106] The test probe consists of an mC-compatible probe and / or an n-mC-compatible probe. The "mC-compatible probe" hybridizes to a base sequence containing a base corresponding to mC at the methylation target site in the detection region of the test nucleic acid (Figure 3(a)). In other words, it is a probe for detecting the detection region where cytosine at the methylation target site is methylated. The "base corresponding to mC" refers to the base (cytosine) corresponding to the mC in the nucleic acid after BS treatment. Since mC is not affected by BS treatment, the base corresponding to mC in this base sequence is cytosine.

[0107] An "n-mC-compatible probe" hybridizes to a base sequence containing a base corresponding to n-mC at the methylation target site in the detection region of the test nucleic acid (Figure 3(b)). In other words, it is a probe for detecting the detection region where the cytosine at the methylation target site is not methylated. On the test nucleic acid, n-mC undergoes cytosine to uracil conversion by BS treatment. Therefore, in nucleic acid amplification products using this as a template, the base corresponding to n-mC in this base sequence becomes thymine.

[0108] When the methylation detection kit of the present invention includes one set of mC-compatible probes and n-mC-compatible probes, the mC-compatible probes and n-mC-compatible probes are configured to hybridize to a nucleotide sequence containing a base corresponding to mC or a nucleotide sequence containing a base corresponding to n-mC at the same methylation target site. In both probes, the nucleotide sequences and base lengths other than the specific cytosine used to detect the presence or absence of methylation may be the same or different. Furthermore, when the kit includes two or more sets of mC-compatible probes and n-mC-compatible probes, in principle, each set is configured to hybridize to a nucleotide sequence containing a base corresponding to mC or a nucleotide sequence containing a base corresponding to n-mC at the same methylation target site.

[0109] The nucleotide sequences of the mC-compatible probes include, for example, nucleotide sequences complementary to the following nucleotide sequences: (a') A nucleotide sequence containing a detection region in which the base corresponding to mC in the detection region is cytosine; (b') A nucleotide sequence in which one or more bases other than the base corresponding to mC in the detection region are added, deleted, and / or substituted in the nucleotide sequence of (a'); (c') A nucleotide sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more sequence identity with the nucleotide sequence of (a'), in which the base corresponding to mC in the detection region is cytosine; or (d') A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of (a').

[0110] Furthermore, if the detection region contains cytosine in addition to the methylation target site, the base sequence of the mC-corresponding probe may also include a base sequence complementary to the base sequence in which the base corresponding to the n-mC is thymine. Specific examples of such base sequences include, for example, the base sequences (a') to (d') in which the base corresponding to the n-mC in the detection region is cytosine.

[0111] Furthermore, as the base sequence of the n-mC-compatible probe, for example, a base sequence that does not hybridize under stringent conditions with a base sequence in which the base corresponding to the n-mC of the methylation target site in the detection region is thymine can be suitably used.

[0112] The nucleotide sequences of n-mC-compatible probes include, for example, nucleotide sequences complementary to the following nucleotide sequences: (a) a nucleotide sequence including a detection region in which the base corresponding to the n-mC of the methylation target site in the detection region is thymine; (b) a nucleotide sequence in which, in the nucleotide sequence of (a), one or more bases other than the base corresponding to the n-mC of the methylation target site in the detection region are added, deleted, and / or substituted; (c) a nucleotide sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more sequence identity with the nucleotide sequence of (a), in which the base corresponding to the n-mC of the methylation target site in the detection region is thymine; or (d) a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of (a).

[0113] Furthermore, if the detection region contains cytosine in addition to the methylation target site, the base sequence of the n-mC-compatible probe may also include a base sequence complementary to the base sequence in which the base corresponding to the n-mC is thymine. Specific examples of such base sequences include, for example, the base sequences in (a) to (d) in which the base corresponding to the n-mC in the detection region other than the methylation target site is cytosine.

[0114] Furthermore, as the base sequence of the n-mC-compatible probe, for example, a base sequence that does not hybridize under stringent conditions with a base sequence in which the base corresponding to the mC of the methylation target site in the detection region is cytosine can be suitably used.

[0115] For a nucleic acid amplification product using the detection region of the test nucleic acid after BS treatment as a template, the mC-compatible probe consists of a base sequence that hybridizes to a base sequence containing the base corresponding to mC, and the n-mC-compatible probe consists of a base sequence that hybridizes to a base sequence containing the base corresponding to n-mC. In this case, the nucleic acid amplification product into which both probes hybridize may be either the sense strand side or the antisense strand side, or both strands. When both probes hybridize to the sense strand side of the nucleic acid amplification product, both probes consist of a base sequence complementary to that sense strand. When both probes hybridize to the antisense strand side of the nucleic acid amplification product, both probes consist of a base sequence complementary to that antisense strand. Furthermore, when both probes hybridize to both the sense strand side and the antisense strand side of the nucleic acid amplification product, the two sets of probes consist of base sequences complementary to the antisense strand and the sense strand of the nucleic acid amplification product, respectively.

[0116] (Test nucleic acid and detection region) The test nucleic acid is usually natural DNA. The specific type is not particularly limited, but examples include genomic DNA, mitochondrial DNA, chloroplast DNA, or fragments thereof (cfDNA, etc.). The test nucleic acid may be the same molecule as the control nucleic acid, a different molecule, or a different type of molecule (DNA).

[0117] The detection region is a region where the presence or absence of methylated cytosine needs to be detected. Therefore, the detection region is a nucleic acid region containing methylated cytosine. The specific type of detection region is not particularly limited, but examples include regions containing CpG sequences, regions containing CpG islands, promoter regions containing CpG sequences, and promoter regions containing CpG islands. Furthermore, for example, the presence or absence of cytosine methylation may be a region related to the presence or absence of disease, the presence or absence of disease risk, the prognosis, the presence or absence of drug efficacy, or the origin of the individual (mother and fetus, etc.).

[0118] While not particularly limited, such regions include, for example, the promoter region, enhancer region, insulator region, intron region, repressor-binding region, untranslated region (UTR) at the 5' or 3' end of a specific gene, and repetitive sequences (Alu sequences, LINE sequences, etc.). Specifically, examples include the promoter regions of the following genes: MLH1, RUNX1, RUNX2, RUNX3, GATA2, GATA4, CEBPA, CEBPB, MAFB, NR4A2, MYOD1, TBX5, Agouti, MeCP2, BDNF, AMPA-type glutamate receptor gene, BRCA1, BRCA2, CDKN2A, GSTP1, MGMT, RASSF1A, APC, VHL, PTEN, E-cadherin gene, TP53, RB1, IGF2, CDH1, DNMT3B, H19, SLIT2, and SOCS1.

[0119] The "MLH1 gene" (MutL homolog 1 gene) is a gene that codes for the MLH1 protein, a type of mismatch repair protein. In humans, it is located on chromosome 3 and is a causative gene for Lynch syndrome-related tumors.

[0120] The "MLH1 gene promoter" is the MLH1 gene expression regulatory region located upstream of the coding region of the MLH1 gene on the genomic DNA. In humans, the 1080-base sequence shown in Sequence ID No. 1 corresponds to the wild-type MLH1 gene promoter.

[0121] In this specification, "methylation of the MLH1 gene promoter" refers to the methylation of specific regions (e.g., C region, D region) in the nucleotide sequence of the MLH1 gene promoter. Methylation of the MLH1 gene promoter results in a suppressed expression of the MLH1 gene. Methylation of the MLH1 gene promoter is also found in cancers such as sporadic colorectal cancer (high-frequency microsatellite instability (MSI-H) colorectal cancer), small intestine cancer, endometrial cancer, ovarian cancer, gastric cancer, renal pelvis and ureteral cancer, pancreatic cancer, biliary tract cancer, brain tumors, Muir-Torre syndrome, malignant melanoma, non-small cell lung cancer, prostate cancer, and thyroid cancer.

[0122] In this specification, the "C region" refers to one of four specific regions present in the nucleotide sequence of the MLH1 gene promoter. In the MLH1 gene promoter nucleotide sequence shown in Sequence ID No. 1, this corresponds to the region consisting of the nucleotide sequence shown in Sequence ID No. 2, corresponding to positions 550 to 621. In the human MLH1 gene promoter, there are eight CpG sequences in the C region.

[0123] In this specification, the "D region" refers to one of the specific regions present in the nucleotide sequence of the MLH1 gene promoter, along with the C region. In the nucleotide sequence of the MLH1 gene promoter shown in SEQ ID NO: 1, this region corresponds to the nucleotide sequence shown in SEQ ID NO: 3, which is from position 689 to 813. In the human MLH1 gene promoter, there are seven CpG sequences in the D region.

[0124] Examples of methylation target sites in the C and / or D regions of the MLH1 gene promoter sequence include, for example, positions 550, 557, 567, 569, 575, 594, 605, and 620 in the C region, and positions 689, 712, 736, 744, 777, 792, and 812 in the D region.

[0125] For example, if the probe targets the methylation target sites of the MLH1 gene, it is sufficient that it be configured to hybridize these methylation target sites with a base sequence containing a base corresponding to a specific cytosine for which the presence or absence of methylation should be detected.

[0126] As a specific example of an MLH1 probe, if the detection region is the C region and the methylation target sites for detecting the presence or absence of methylation are the cytosines at positions 557, 567, and 569 in the MLH1 gene promoter sequence shown in Sequence ID No. 1, then an MLH1-mC corresponding probe could be, for example, the 27-base polynucleotide shown in Sequence ID No. 4. Furthermore, an MLH1-n-mC corresponding probe that pairs with the MLH1-mC corresponding probe could be, for example, the 20-base polynucleotide shown in Sequence ID No. 5.

[0127] Furthermore, if the detection region is the D region and the methylation target sites for detecting the presence or absence of methylation are the cytosines at positions 736 and 744 in the MLH1 gene promoter sequence shown in Sequence ID No. 1, then an example of an MLH1-mC corresponding probe is a 19-base polynucleotide shown in Sequence ID No. 6. In addition, an example of a 16-base polynucleotide shown in Sequence ID No. 7 is a MLH1-n-mC corresponding probe that pairs with the MLH1-mC corresponding probe.

[0128] (8) Test primer set The "test primer set" consists of one or more primers configured to hybridize to the base sequence of a detection region containing a methylation target site in the nucleic acid under test, and to amplify the said detection region.

[0129] The test primer set only needs to be capable of amplifying the detection region described above for the test probe, and its specific design and configuration are not particularly limited. The amplified sequence may include any sequence in addition to the detection region.

[0130] The basic design and configuration (including labeling with a labeling substance) of the Fw primer and Rv primer of the test primer set are the same as those of the Fw primer and Rv primer of the control primer set, so a detailed explanation is omitted here.

[0131] If the detection region is a sequence region that includes all or part of the C and / or D regions of the MLH1 gene promoter, the cytosine in the CpG sequence, which is the methylation target site, is located at eight positions in the C region (positions 550, 557, 567, 569, 575, 594, 605, and 620) and seven positions in the D region (positions 689, 712, 736, 744, 777, 792, and 812) in the MLH1 gene promoter sequence shown in Sequence ID No. 1. Therefore, a primer set for amplifying these regions only needs to be able to amplify a sequence region containing at least one of these methylation target sites as the detection region.

[0132] The detection region may be one or more. In the case of the MLH1 gene promoter, for example, the detection region may be a single nucleotide sequence containing all or part of the C and D regions, or it may be two nucleotide sequences containing all or part of the C region and all or part of the D region.

[0133] The base length of the region amplified by a pair of primer sets is not particularly limited as long as it is long enough to amplify the detection region. For example, in the case of the MLH1 gene promoter, the C region is a 72-base sequence shown in SEQ ID NO: 2, the D region is a 125-base sequence shown in SEQ ID NO: 3, and the MLH1 gene promoter is a 1080-base sequence shown in SEQ ID NO: 1. Therefore, for example, if the detection region is a base sequence containing only a portion of the methylation target sites in the C region, it only needs to be 40 bases or longer, and if the entire MLH1 gene promoter is used as the detection region, it only needs to be at least 1080 bases long.

[0134] For example, if the D region of the MLH1 gene promoter sequence is used as the detection region, and the cytosines at positions 736 and 744 in the sequence shown in Sequence ID No. 1 are used as the target methylation sites, the forward (Fw) primer can be designed based on the description of the control primer set, with the cytosine at position 736 as the methylation target site at the 3' end of the detection region. Similarly, the reverse (Rv) primer can be designed based on the description of the control primer set, with the cytosine at position 736 as the methylation target site at the 3' end of the detection region.

[0135] As a specific example of a test primer set, if the detection region is the MLH1 gene promoter, for example, a 25-base polynucleotide shown in SEQ ID NO: 12 can be used as the Fw primer and a 27-base polynucleotide shown in SEQ ID NO: 13 can be used as the Rv primer for amplification of the MLH1 C region. In this case, the detection region is the C region, and the methylation target sites for detecting the presence or absence of methylation are the cytosines at positions 557, 567, and 569 in the MLH1 gene promoter sequence shown in SEQ ID NO: 1.

[0136] Furthermore, if the detection region is the D region, and the methylation target sites for detecting the presence or absence of methylation are the cytosines at positions 736 and 744 of the MLH1 gene promoter sequence shown in SEQ ID NO: 1, then, for example, a 24-base polynucleotide shown in SEQ ID NO: 14 can be used as the Fw primer and a 23-base polynucleotide shown in SEQ ID NO: 15 can be used as the Rv primer for amplifying the MLH1 D region.

[0137] 1-4. Applications The accuracy determination kit and device of this embodiment can be used to assist in diagnoses that require a judgment based on the presence or absence of cytosine methylation in the detection region, and may be provided as a diagnostic assistance kit and a diagnostic assistance device. For example, the presence or absence of cytosine methylation can be used to assist in the diagnosis of the presence or absence of disease, the presence or absence of risk of disease, the prognosis, the presence or absence of drug efficacy, and the originating individual (mother and fetus, etc.). In this case, preferably, the diagnostic assistance kit and device include a test probe and / or a test primer.

[0138] There are no particular limitations on the diseases for which the presence or absence of the disease, the presence or absence of risk of the disease, the prognosis, and the presence or absence of drug efficacy are determined. Specific diseases include, for example, cancer, lifestyle-related diseases, neurological disorders, mental disorders, autoimmune diseases, allergic diseases, inflammatory diseases, infertility, and congenital abnormalities. Specific cancers include, for example, Lynch syndrome-related tumors, colorectal cancer, endometrial cancer, ovarian cancer, small intestine cancer, renal pelvis and ureteral cancer, gastric cancer, esophageal cancer, hematopoietic malignancies, brain tumors, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, breast cancer, lung cancer, and sebaceous adenomas and keratocaneurycotas associated with Moore-Tre syndrome. Specific lifestyle-related diseases include, for example, smoking, hyperlipidemia, hypertension, and metabolic syndrome. Specific neurological disorders include, for example, Alzheimer's disease and Parkinson's disease. Specific mental disorders include, for example, Rett syndrome, schizophrenia, depression, and bipolar disorder. Specific autoimmune diseases include, for example, rheumatoid arthritis and systemic lupus erythematosus. Specific inflammatory diseases include, for example, hepatitis and gastritis. Specific infertility conditions include, for example, infertility due to ovulation disorders, infertility due to fallopian tube disorders, and infertility due to sperm abnormalities. Specific congenital abnormalities include ICF syndrome and various other imprinting disorders.

[0139] 2. Method for Determining the Accuracy of Bisulfite Treatment 2-1. Overview A second aspect of the present invention is a method for determining the accuracy of bisulfite treatment (often abbreviated as "accuracy determination method" in this specification). According to the accuracy determination method of the present invention, it is possible to determine whether the bisulfite treatment is good or bad.

[0140] 2-2. Process A flowchart of the accuracy determination method of the present invention is shown in Figure 4. As shown in this figure, the accuracy determination method of the present invention includes the control bisulfite treatment step (S0102), the control probe binding step (S0104), the control probe measurement step (S0105), and the bisulfite treatment determination step (S0106) as essential steps, and the nucleic acid preparation step (S0101) and the control region amplification step (S0103) as optional steps. Each step will be described below.

[0141] (1) Nucleic Acid Preparation Step The "Nucleic Acid Preparation Step" (S0101) is a step in which the nucleic acid to be used for determination by the method of this embodiment is prepared. In this embodiment, the control nucleic acid is the subject of determination, so the control nucleic acid is prepared by this step. The control nucleic acid has been described in detail in the first embodiment, so its explanation is omitted here.

[0142] If the control nucleic acid is a nucleic acid derived from the test subject, the specific preparation method shall be in accordance with the description of the nucleic acid preparation step in the third embodiment.

[0143] If the control nucleic acid is not derived from the subject, it can be prepared using any method known in the art. The preparation method can be appropriately selected according to the purpose, such as the origin of the control nucleic acid, and is not particularly limited. For example, it can be prepared by known genetic engineering techniques (Sambrook et al., Molecular Cloning, 2nd edition, Current Protocols in Molecular Biology (1989), Cold Spring Harbor Laboratory Press; Ausubel et al., Short Protocols in Molecular Biology, 3rd edition, A compendium of Methods from Current Protocols in Molecular Biology (1995), John Wiley & Sons, etc.), conventional methods using commercially available nucleic acid synthesizers, nucleic acid amplification methods, screening from cDNA libraries, or a combination thereof.

[0144] The prepared nucleic acids can be used as is, or subjected to additional processing such as purification as appropriate, before being used in the next control bisulfite treatment step.

[0145] (2) Control Bisulfite Treatment Step The "Control Bisulfite (BS) Treatment Step" (S0102) is a step in which a control nucleic acid is treated with BS. In this step, the entire sample containing the control nucleic acid may be treated, or only a portion of it may be treated. For example, in this step, a portion of the sample containing the control nucleic acid may be subjected to BS treatment, and the remaining portion may be saved as untreated detection nucleic acid for subsequent steps.

[0146] BS treatment can be carried out using conventional methods in the field. For example, the genomic DNA and bisulfite can be mixed and reacted at a predetermined temperature for a predetermined time. The bisulfite is not limited, but for example, sodium bisulfite (NaHSO3), potassium bisulfite (KHSO3), ammonium bisulfite ((NH4)HSO4), etc. can be used. Sodium bisulfite is preferred. The concentration of bisulfite in the reaction solution used for BS treatment is not particularly limited as long as it is sufficient to convert unmethylated cytosine in the genomic DNA. For example, a final concentration of 1M to 15M, 2M to 10M, or 3M to 6M is acceptable. The reaction conditions, such as reaction temperature and reaction time, and the incubation conditions (temperature and time) can be appropriately set according to the amount of bisulfite added. For example, if bisulfite is added at a final concentration of 6M, incubation at 50 to 80°C for 10 minutes to 7 hours is sufficient.

[0147] In the reaction after BS treatment, washing may be performed to remove bisulfites if necessary.

[0148] In this process, if the BS treatment is successful, all n-mC molecules in the control region will be replaced with uracil.

[0149] (3) Control Region Amplification Step The "Control Region Amplification Step" (S0103) is a step in which the control region is amplified by nucleic acid amplification using a control primer set capable of amplifying the control region, with the control nucleic acid after the control BS step as a template.

[0150] For the control primer set, you may use a primer set having the configuration described in "(3) Control Primer Set" in the chapter "1-3. Configuration" of the first embodiment.

[0151] The method for amplifying the control region is achieved by nucleic acid amplification. "Nucleic acid amplification" refers to a method of amplifying a specific target nucleic acid region sandwiched between Fw / Rv primers by repeatedly performing a nucleic acid extension reaction with nucleic acid polymerase. Examples include PCR (polymerase chain reaction), LAMP (Loop-Mediated Isothermal Amplification), and ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids). PCR is preferred. This is because it is the most widely used method in this field, with a wide range of reagents, kits, and reaction equipment available, as well as various application technologies that have been developed and published. For example, qPCR (quantitative PCR) is known as a quantitative nucleic acid amplification method using PCR, and various dedicated reagents, kits, and reaction equipment for qPCR are commercially available from various manufacturers.

[0152] PCR reaction conditions vary depending on the base length of the control region to be amplified, the amount of template genomic DNA (non-BS treated genomic DNA and BS treated genomic DNA), the base length and Tm value of the control primers used, the optimal reaction temperature of the nucleic acid polymerase used, and the optimal pH. Therefore, these conditions should be considered and determined appropriately. As an example, a denaturation reaction can be performed at 94-95°C for 5 seconds to 5 minutes, an annealing reaction at 50-70°C for 10 seconds to 1 minute, and an extension reaction at 68-72°C for 30 seconds to 3 minutes. This constitutes one cycle, and this can be repeated for 15-40 cycles, with a final extension reaction at 68-72°C for 30 seconds to 10 minutes. When using a commercially available PCR kit, in principle, the protocol provided with the kit should be followed.

[0153] In quantitative nucleic acid amplification reactions, it is desirable to label the amplified product so that it can be identified. Methods for labeling the amplified product are not particularly limited, but include, for example, pre-labeling primers, using labeled nucleotides as substrates in the nucleic acid amplification reaction, and pre-labeling probes that specifically bind to the amplified product. The labeling substance is not particularly limited, but radioactive isotopes, fluorescent substances, or organic compounds such as DIG or biotin can be used.

[0154] To confirm the presence or absence of control nucleic acids and the suitability of the hybridization conditions, control nucleic acids containing control regions that have not undergone BS treatment may be additionally amplified. In this case, the content of the amplification step (non-BS treated control region amplification step) shall be the same as described above for this step. The amplified product obtained by the non-BS treated control region amplification step shall be called the non-BS treated control region amplified product (n-BS treated control region amplified product).

[0155] The control region amplification step and the non-BS processed control region amplification step can be performed simultaneously or separately. The amplification reaction conditions for the control region amplification step and the non-BS processed control region amplification step may be the same or different.

[0156] (4) Control probe coupling process The "control probe coupling process" (S0104) is a process of hybridizing a control probe, which is capable of being coupled to the control region and consists of a BS-processed detection probe and a non-BS-processed detection probe, to the control region after the control BS process.

[0157] The control region into which each probe is hybridized in this process may be the control region in the control nucleic acid or the control region in its amplification product. When hybridizing to the control region in the amplification product, this process is performed simultaneously with or after the control region amplification process (S0103).

[0158] The control probes include a BS-treated detection probe and an n-BS-treated detection probe. Both probes may be applied simultaneously to the control nucleic acid after the control BS process to allow hybridization, or a portion of the control nucleic acid after the control BS process may be treated with the BS-treated detection probe and hybridized, while the remaining portion of the control nucleic acid after the control BS process may be treated with the n-BS-treated detection probe and hybridized, or the reverse order may be used.

[0159] Each control probe may be one having the configuration described in "(1) Bisulfite-treated detection probe (BS-treated detection probe)" and "(5) Non-bisulfite-treated detection probe (n-BS-treated detection probe)" in the chapter "1-3. Configuration" of the first embodiment.

[0160] The reaction conditions for the hybridization reaction in which a control probe is bound to the amplified product are not particularly limited as long as conditions are in which nucleic acids having a base sequence complementary to the control region can be bound to the control region, but it is preferable to carry out the reaction under stringent conditions.

[0161] In the BS-treated control region amplification product obtained after the control region amplification step (S0103) of the BS-treated control nucleic acid, if the BS treatment was successful, the base corresponding to n-mC in the control region is substituted with T. In this case, a BS-treated detection probe having a base sequence complementary to the base sequence of the control region after the base substitution can specifically bind, while an n-BS-treated detection probe cannot bind (Figure 2(a)). On the other hand, if the BS treatment failed, the base corresponding to n-mC in the control region remains C. Therefore, in this case, the BS-treated detection probe cannot bind, and an n-BS-treated detection probe having a base sequence complementary to the base sequence of the untreated control region can specifically bind (Figure 2(b)).

[0162] As described above, in this process, the quality of the BS treatment in the BS treatment process (S0102) is converted into the difference in the binding amount of each control probe.

[0163] To confirm the presence or absence of control nucleic acids and the suitability of the hybridization conditions, a control probe may be bound to an additional n-BS-treated control region amplification product (n-BS-treated control probe binding step).

[0164] (5) Control probe measurement process The "control probe measurement process" (S0105) is a process of measuring the amount of each probe bonded after the control probe bonding process (S0104).

[0165] In the control probe binding step (S0104), the control probe was bound to the control nucleic acid or its amplified product by a hybridization reaction. Several methods with different detection means are known for measuring the amount of probe bound based on the hybridization reaction. Examples include Southern blot hybridization, microarray method, surface plasmon resonance method, or quartz crystal microbalance method.

[0166] A. Southern Blot Hybridization Method The Southern Blot Hybridization Method is a method in which a control nucleic acid containing a control region or its amplified product (in this specification, a BS-treated control region amplified product or an n-BS-treated control region amplified product) is separated based on base length size by electrophoresis using an agarose gel or polyacrylamide gel, etc., and after blotting onto a filter, it is detected using a probe having a base sequence specific to the target nucleic acid (in this specification, a control region amplified product containing n-mC).

[0167] The control probe may be pre-labeled with various labeling substances as described in "(1) Bisulfite-treated detection probe (BS-treated detection probe)" in the "1-3. Composition" section of the first embodiment. This allows for the measurement of the amount of target nucleic acid bound to the DNA amplification product. Alternatively, the primer may be pre-labeled, and the amount of binding between the amplification product containing the labeling substance and the control probe may be measured.

[0168] The amount of binding between the probe based on the label and the amplified product can be measured using measuring devices such as a fluorescence scanner, chemiluminescence imaging analyzer, imaging analyzer, or scintillation counter. For example, when a fluorescent substance is used as the label, the amount of binding can be obtained as a measured value by detecting the fluorescence signal using a fluorescence scanner and quantifying the signal intensity using image analysis software.

[0169] In this specification, "measured value" refers to a value indicating the amount of each probe bound to the target nucleic acid amplification product, as measured in this process and in the test dose measurement process (S0223) described later. The measured value may be an absolute value such as volume, weight, or number of probes, or it may be a relative value such as concentration, ionic strength, absorbance, or fluorescence intensity.

[0170] B. Microarray Method The "microarray method," as explained in "(6) Microarray" in the chapter "1-3. Configuration" of the first aspect, involves densely arranging and immobilizing probes having a base sequence complementary to all or part of the base sequence of the target nucleic acid on a substrate in a spot-like manner, reacting a sample containing the target nucleic acid with these probes, and detecting and measuring the nucleic acid hybridized to the spots using fluorescence or the like.

[0171] The detection and quantification of target nucleic acids can be performed in essentially the same manner as the Southern blot hybridization method described above. For example, by pre-labeling primers, an amplified product containing the labeling substance can be obtained. This amplified product specifically binds to a fixed probe, and the label on the amplified product can be detected and measured using a microplate reader or fluorescence scanner, thereby achieving detection of the target nucleic acid. Furthermore, by quantifying the labeling intensity using image analysis software, the amount of binding between the control probe and the amplified product can be obtained as a measured value.

[0172] C. Surface Plasmon Resonance Method The "Surface Plasmon Resonance (SPR) method" is a method for highly sensitively detecting and quantifying adsorbents on the surface of a metal thin film by utilizing the surface plasmon resonance phenomenon, in which the reflected light intensity is significantly attenuated at a specific incident angle (resonance angle) when the incident angle of laser light irradiated onto a metal thin film is changed. In this invention, for example, a control probe is immobilized on the surface of the metal thin film, and other parts of the metal thin film surface are blocked. Then, a control nucleic acid containing a control region or its amplified product (in this specification, a BS-treated control region amplified product or an n-BS-treated control region amplified product) is passed over the metal thin film surface to capture the amplified product that has specifically bound to the control probe via base pairing. A hybrid of the control probe and the DNA amplified product can be detected and measured from the difference in measurement values ​​before and after sample flow. Measurement by surface plasmon resonance can be performed, for example, using an SPR sensor commercially available from Biacore.

[0173] D. Quartz Crystal Microbalance Method The "Quartz Crystal Microbalance (QCM) method" is a mass measurement method that quantitatively detects minute amounts of adsorbed material by measuring the change in resonance frequency, utilizing the phenomenon that when a substance is adsorbed onto the electrode surface attached to a quartz crystal, the resonance frequency of the quartz crystal decreases in proportion to the mass of the adsorbed substance. Similar to the SPR method, detection and quantification using this method can be performed using a commercially available QCM sensor, for example, by base pairing with a control probe fixed to the electrode surface to detect and measure the control region in the amplification product after the control region amplification step (S0103) or a control nucleic acid.

[0174] Various nucleic acid quantification kits based on the above methods are commercially available from various life science manufacturers and can also be used.

[0175] (6) Bisulfite Treatment Determination Step The "bisulfite (BS) treatment determination step" (S0106) is a step in which the quality of the BS treatment in the BS treatment step (S0102) (whether or not the BS reaction was normal) is determined based on the binding ratio of the BS treatment detection probe and the n-BS treatment detection probe after the control probe measurement step (S0105).

[0176] The method for calculating the binding ratio is not particularly limited, as long as it yields a value that reflects the difference between the binding amount of the BS-processed detection probe and the binding amount of the n-BS-processed detection probe. For example, a value based on the quotient or reciprocal thereof of the binding amount of the n-BS-processed detection probe and the binding amount of the BS-processed detection probe can be calculated. In this case, additional calculations can be performed. The specific content of the additional calculations is not particularly limited. For example, in addition to arithmetic operations with any number (including the binding amounts of other probes), it can be an exponent of this ratio, a logarithm of this ratio, a power, a root, or a combination thereof. For example, the calculations may be performed independently on the numerator and / or denominator, or on the whole.

[0177] While the determination method based on the binding ratio is not limited, for example, a determination value can be calculated using a BS processing determination formula, and the determination can be made based on that value.

[0178] The aforementioned BS processing determination formula can be expressed, for example, by the following formula: (BS processing determination formula) BS processing determination value = n-BS processing detection probe coupling amount / (n-BS processing detection probe coupling amount + BS processing detection probe coupling amount)

[0179] In the above formula, "BS-treated detection probe binding amount" is a measured value indicating the binding amount of the BS-treated detection probe to the BS-treated control region obtained in the control probe measurement step (S0105). In the above formula, "n-BS-treated detection probe binding amount" is a measured value indicating the binding amount of the n-BS-treated detection probe to the BS-treated control region obtained in the control probe measurement step (S0105).

[0180] According to this BS processing judgment formula, a large judgment value (close to 1) is obtained if the BS processing is poor, and a small judgment value (close to 0) is obtained if the BS processing is good.

[0181] By substituting the respective measured values ​​into the BS processing judgment formula, a judgment value can be calculated. Based on the obtained judgment value, the quality of the BS processing in the BS processing step (S0102) can be determined.

[0182] The determination method based on the judgment value is not limited to this method, but examples include a method in which a cutoff value is set in advance and the quality of the BS treatment is determined based on that cutoff value, and a method in which the quality of the BS treatment is determined based on the statistically significant difference when comparing the judgment value obtained in this process with the judgment value calculated based on a standard sample in which the BS treatment was measured in advance and was good.

[0183] A "cutoff value" is a boundary value used to classify quantitative results into two groups. For example, a predetermined value can be set as the cutoff value. If the judgment value is greater than or equal to that value, it can be determined that there is a high probability that the BS processing in the BS processing step (S0102) is poor. Conversely, if the judgment value is lower than that value, it can be determined that there is a high probability that the BS processing in the BS processing step (S0102) is good.

[0184] There are no particular limitations on the method for setting the cutoff value. The cutoff value may be determined based on values ​​measured in the same control region, or it may be determined based on a value expected from the number of methylated and unmethylated cytosines in the control region. For example, based on the relationship between the proportion of methylated nucleic acids and the judgment value derived by a similar method, the group of judgment values ​​can be classified by percentile, and the percentile value used for that classification can be set as the cutoff value. Specifically, for example, if the 5th percentile of the judgment value is set as the cutoff value in advance, and values ​​above that value are defined as poor and values ​​below that value are defined as good, then if the judgment value calculated by the newly implemented methylation detection method is less than the 5th percentile, it can be determined that the BS treatment in the BS treatment step (S0102) of that method is likely to be good.

[0185] The method used to determine "statistical significance" is not limited to any known test method capable of determining significance. Examples include the t-test and multiple comparison tests. "Statistically significant" means that, under the null hypothesis, the randomness of the obtained value is statistically low and it is meaningful, while "significant difference" means that there is a significant difference between the two values. Specifically, this includes cases where the p-value is smaller than the significance level, such as 5%, 1%, 0.3%, 0.2%, or 0.1%.

[0186] In this process, if the average judgment value calculated based on standard samples in which BS treatment was successful and the judgment value of the test sample are statistically processed using the t-test method or the like, and there is no statistically significant difference between the two, then it can be determined that the BS treatment in the BS treatment process (S0102) was successful. On the other hand, if a significant difference is found, it can be determined that there is a high possibility that the BS treatment was unsuccessful. "Average judgment value calculated based on standard samples in which BS treatment was successful" refers to the average value of judgment values ​​obtained from multiple standard samples in which BS treatment was confirmed to be successful using the same methylation detection method performed on the test sample.

[0187] The necessity of performing this step may be determined based on other indicators. For example, if an n-BS-treated control probe binding step is performed to confirm the presence or absence of control nucleic acids or the suitability of hybridization conditions, this step may be performed only if the binding value obtained from the results is above a certain level.

[0188] If an effective amount of control nucleic acid is present and the probe sequence and hybridization conditions are appropriate, all unmethylated cytosines in the control nucleic acid will be converted to uridine, and only the BS-treated detection probe should be able to bind, while the n-BS-treated detection probe should not. Therefore, theoretically, the judgment value obtained from the n-BS-treated control probe binding step should be 0 based on the above judgment formula.

[0189] In this way, the suitability of the reaction conditions can be further determined based on the judgment value obtained from the results of the n-BS treatment control probe coupling step.

[0190] 3. Methylation Detection Method 3-1. Overview A third aspect of the present invention is a method for detecting methylation in the detection region of a test nucleic acid (often abbreviated as "methylation detection method" in this specification). According to the methylation detection method of the present invention, when determining whether or not methylation is present in the detection region of a test nucleic acid, it is possible to determine the quality of the bisulfite treatment used for methylation detection. This makes it possible to provide a determination result with high accuracy and low false-identification regarding the presence or absence of methylation in the detection region of the test nucleic acid.

[0191] 3-2. Process Flowcharts of the methylation detection method of the present invention are shown in Figures 5 to 7. The flowchart shown in Figure 5 is the main flowchart of the methylation detection method of the present invention, and includes the bisulfite treatment step (S0202), quantitative nucleic acid amplification step (S0203), bisulfite treatment determination step (S0204), and methylation determination step (S0205) as essential steps, and the nucleic acid preparation step (S0201) as an optional step.

[0192] Furthermore, within the quantitative nucleic acid amplification step (S0203) in the main flow shown in Figure 5, two subflows shown in Figures 6 and 7 are processed in parallel. The flow shown in Figure 6 is a flow for performing quantitative nucleic acid amplification on a control region (control region flow), and includes a control probe binding step (S0212) and a control probe measurement step (S0213) as essential steps, and includes a control region amplification step (S0211) as an optional step. The flow shown in Figure 7 is a flow for performing quantitative nucleic acid amplification on a detection region (detection region flow), and includes a detection region amplification step (S0221), a test probe binding step (S0222), and a test dose measurement step (S0223) as essential steps.

[0193] The control region flow and the detection region flow are independent flows, and their processing order is not restricted. For example, the control region flow may be performed before the detection region flow, or vice versa. Furthermore, both may be performed simultaneously. In particular, steps with similar operational procedures in both flows can be performed simultaneously. That is, the control region amplification step (S0211) and the detection region amplification step (S0221) for nucleic acid amplification, the control probe binding step (S0212) and the test probe binding step (S0222) for probe binding, and the control probe measurement step (S0213) and the test dose measurement step (S0223) for probe measurement may be performed simultaneously in a single reaction system (in a single sample) using the primer sets and probes used in each step. The following describes each step in detail.

[0194] (1) Nucleic acid preparation process The "nucleic acid preparation process" (S0201) is a process for preparing the test nucleic acid from a sample derived from the test subject.

[0195] In this specification, "subject" refers to an individual subjected to the methylation detection method of the present invention. While not limited to this type, it is generally human. For example, a patient suspected of having the disease being tested is preferred.

[0196] The type of sample is not restricted. Blood, such as whole blood, is preferred due to its easy availability regardless of the time of collection, low invasiveness to the subject, and ease of handling as a liquid. Other samples may include formalin-fixed paraffin-embedded sections and frozen tissue. ctDNA (circulating tumor DNA) and cfDNA (cell-free DNA) can also be used as samples.

[0197] If the sample is blood, it is acceptable as long as the blood has been collected according to a known method, and there are no particular limitations. For example, blood collected directly by injection from a vein, blood that has been treated with anticoagulation by adding heparin or the like to collected whole blood, and blood that has been stored refrigerated or frozen can all be used as samples.

[0198] The sample volume to be used can be 100 μL to 1 mL, 50 μL to 5 mL, 20 μL to 10 mL, 10 μL to 50 mL, 5 μL to 80 mL, or 1 μL to 100 mL if it is blood.

[0199] The method for extracting DNA from a sample should be prepared according to the standard methods in the relevant field, depending on the type of sample, such as blood or tissue. For example, extraction can be performed according to the nucleic acid extraction method (DNA extraction method) described in Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Furthermore, various life science manufacturers commercially offer kits for extracting nucleic acids from various samples, which may also be used. When using a kit, the specific extraction method should be carried out according to or in accordance with the attached protocol.

[0200] (2) Bisulfite Treatment Process The "Bisulfite (BS) Treatment Process" (S0202) is a process in which the test nucleic acid and the control nucleic acid are treated with BS. In this process, a portion of the sample containing genomic DNA is subjected to BS treatment, and the remaining portion is stored as untreated genomic DNA for subsequent processes.

[0201] This process is similar to the description of the control bisulfite treatment process (S0102) in the second embodiment. Only the differences from the control bisulfite treatment process will be described below.

[0202] In this process, both the test nucleic acid and the control nucleic acid are subjected to BS treatment simultaneously in the same processing environment.

[0203] In this step, a test nucleic acid containing the detection region, such as genomic DNA derived from the subject, is used. The control nucleic acid may be a molecule other than the test nucleic acid, the same molecule as the test nucleic acid, or an exogenous nucleic acid molecule. If the control nucleic acid is an exogenous nucleic acid molecule or other nucleic acid molecule that needs to be added from an external source to the sample containing the test nucleic acid, in this specification, the control nucleic acid is mixed with the test nucleic acid before performing this step.

[0204] (3) Quantitative nucleic acid amplification step The "quantitative nucleic acid amplification step" (S0203) is a step in which a quantitative nucleic acid amplification reaction is performed using the BS-treated genomic DNA after the BS treatment step and the non-BS-treated genomic DNA, i.e., the genomic DNA before the BS treatment step.

[0205] "Quantitative nucleic acid amplification reaction" refers to the quantitative measurement of the amplified product (amplicon) produced by the nucleic acid amplification reaction. The nucleic acid amplification reaction is described in accordance with the description in the control region amplification step (S0103) of the second embodiment.

[0206] In this process, as mentioned above, two independent subflows, namely the control region flow shown in Figure 6 and the detection region flow shown in Figure 7, are processed in parallel. Each subflow will be explained below.

[0207] (3-1) Control Region Flow The control region flow is a flow in which a quantitative nucleic acid amplification reaction is performed on the control region using BS-treated control nucleic acid and non-BS-treated control nucleic acid as templates. This flow includes a control region amplification step (S0211), a control probe binding step (S0212), and a control probe measurement step (S0213). Each step will be explained below.

[0208] (3-1-1) Control Region Amplification Step The "Control Region Amplification Step" (S0211) is a step in which the control region is amplified by nucleic acid amplification using a control primer set capable of amplifying the control region, with the control nucleic acid after the bisulfite treatment step (S0202) as a template.

[0209] The specific details of this process are the same as those described in the control region amplification process (S0103) in the second embodiment.

[0210] Furthermore, the basic conditions for the nucleic acid amplification reaction in this step and the detection region amplification step (S0221) in the detection region flow described later may be the same, except for the difference in the primer sets used. Therefore, although both steps constitute independent flows, they can be carried out in the same reaction system. In that case, in the test section using the control nucleic acid of this step as a template, the nucleic acid amplification reaction can be carried out simultaneously using the primer set having the configuration described in "(3) Control Primer Set" in the "1-3. Configuration" section and the primer set having the configuration described in "(8) Test Primer Set".

[0211] (3-1-2) Control probe binding step The "control probe binding step" (S0212) is a step in which a control probe, which consists of a BS-treated detection probe and a non-BS-treated detection probe and is capable of binding to the control region in the control nucleic acid, is hybridized to the control region after the bisulfite treatment step (S0202). This step is in accordance with the description of the control probe binding step (S0104) in the second embodiment.

[0212] Although this step and the test probe coupling step (S0222) in the detection region flow described later constitute independent flows, the probe coupling conditions in both steps may be the same, except for the difference in the probes used. Therefore, both steps can be performed in the same reaction system. For example, as described in the control region amplification step (S0211), if the control region amplification step (S0211) and the detection region amplification step (S0221) in the detection region flow described later are performed in the same reaction system, then this step and the test probe coupling step (S0222) can then be performed in the same reaction system. In that case, when performing this step, two types of probes having the configuration described in "(7) Test Probe" in the "1-3. Configuration" section of the first embodiment, namely the mC-compatible probe and the n-mC-compatible probe, should be added simultaneously to the test section to which the BS-treated detection probe is coupled to the BS-treated control region amplification product.

[0213] (3-1-3) Control probe measurement process The "control probe measurement process" (S0213) is a process of measuring the amount of each probe bonded after the control probe bonding process (S0212). This process is in accordance with the description of the control probe measurement process (S0105) in the second embodiment.

[0214] Although this process and the sample quantity measurement process (S0223) in the detection region flow described later constitute independent flows, the measurement conditions in both processes may be the same as long as the binding of the amplified product to the probe in each process can be identified. Therefore, both processes can be carried out in the same reaction system. For example, by using different labeling substances in each flow, the amount of binding between the amplified product and the probe in each flow can be measured based on the difference in labeling substances, even when carried out in the same reaction system.

[0215] (3-2) Detection Region Flow The detection region flow is a flow in which a quantitative nucleic acid amplification reaction is performed on the detection region using BS-treated genomic DNA as a template. This flow includes a detection region amplification step (S0221), a test probe binding step (S0222), and a test dose measurement step (S0223). Each step will be explained below.

[0216] (3-2-1) Detection Region Amplification Step The "detection region amplification step" (S0221) is a step in which the detection region is amplified using the test nucleic acid after the BS treatment step as a template, using a test primer set capable of amplifying the detection region. This step may be performed simultaneously with the control region amplification step (S0211) or independently. Preferably, this step is performed simultaneously with the control region amplification step (S0211). In this case, as described above, this step and the control region amplification step (S0211) in the control region flow described above can be performed in the same reaction system.

[0217] The test primer set may be a primer set having the configuration described in "(8) Test Primer Set" in the chapter "1-3. Configuration" of the first embodiment.

[0218] The detection region is amplified by nucleic acid amplification. The nucleic acid amplification method and its reaction conditions may be carried out in accordance with the control region amplification step (S0103). This step may be performed only if the result of the BS processing judgment step (S0204) described later is judged as good.

[0219] (3-2-2) Test probe coupling process The "test probe coupling process" (S0222) is a process in which an mC-compatible probe and a non-mC-compatible probe are hybridized to the amplified product after the detection region amplification process.

[0220] The contents of this step may be carried out in accordance with the control probe binding step (S0104). This step may be carried out simultaneously with the control probe binding step (S0212) or independently. Preferably, this step is carried out simultaneously with the control probe binding step (S0212). In this case, as described above, this step and the control probe binding step (S0212) in the control region flow described above can be carried out in the same reaction system.

[0221] The test probe consists of an mC-compatible probe and an n-mC-compatible probe, as described in "(7) Test Probe" in the chapter "1-3. Composition". The mC-compatible probe specifically hybridizes to a base sequence containing a base corresponding to mC at the methylation target site in the detection region of the test nucleic acid (Figure 3(a)), and the n-mC-compatible probe specifically hybridizes to a base sequence containing a base corresponding to n-mC at the methylation target site in the detection region of the test nucleic acid (Figure 3(b)).

[0222] The test probes used in this process may be two types of probes (mC-compatible probe and n-mC-compatible probe) having the configuration described in "(7) Test Probes" in the chapter "1-3. Configuration". Either probe will be used in this process. This process may be performed only if the result of the BS processing judgment process (S0204) described later is judged as good.

[0223] (3-2-3) Test dose measurement process The "Test dose measurement process" (S0223) is a process for measuring the amount of each probe bound after the test probe binding process (S0222).

[0224] In the test probe binding step (S0222), similar to the control probe binding step (S0104), each test probe is bound to the detection region amplification product by a hybridization reaction. In this step as well, the amount of probe bound based on the hybridization reaction can be measured using methods such as Southern blot hybridization, microarray method, surface plasmon resonance method, or quartz crystal microbalance method, based on the hybridization reaction described in the measurement step (S0105) of the second embodiment. Therefore, this step can also be basically measured using a method similar to that of the measurement step (S0105).

[0225] This step may be performed simultaneously with the control probe measurement step (S0213) or independently. Preferably, this step is performed simultaneously with the control probe measurement step (S0213). In this case, as described above, this step and the control probe measurement step (S0213) in the control region flow can be performed in the same reaction system. This step may be performed only if the result of the BS treatment judgment step (S0204) described later is judged as good.

[0226] (4) Bisulfite Treatment Determination Step The "bisulfite (BS) treatment determination step" (S0204) is a step that determines whether the BS treatment in the bisulfite treatment step (S0202) was good or bad (whether the BS reaction was normal or not) based on the ratio of the amount of BS treatment detection probe bound to the amount of non-BS treatment detection probe bound to this bisulfite treatment step (S0202). This step is in accordance with the description of the bisulfite treatment determination step (S0106) in the second embodiment.

[0227] (5) Methylation determination step The "methylation determination step" (S0205) is a step performed after the test amount measurement step (S0223) to determine whether or not methylation is present in the detection region based on the ratio of the amount bound by the mC-compatible probe to the amount bound by the non-mC-compatible probe.

[0228] This process is performed only if the result of the BS processing judgment process (S0204) is judged as good (the BS reaction was normal), and is generally performed after the BS processing judgment process (S0204). This is because if the result of the BS processing judgment process (S0204) is judged as bad, the reliability of the judgment result in this process is lost, making it meaningless. This process alone may be performed only if the result of the BS processing judgment process (S0204) is judged as good (the BS reaction was normal), or in addition to this process, the detection region amplification process (S0221), the test probe coupling process (S0222), the test amount measurement process (S0223), etc. may also be performed only if the result of the BS processing judgment process (S0204) is judged as good (the BS reaction was normal) (Figure 5).

[0229] The method for calculating the binding amount ratio is not particularly limited, as long as it yields a value that reflects the difference between the binding amount of the mC-compatible probe and the binding amount of the n-mC-compatible probe. For example, a value based on the quotient or reciprocal thereof of the binding amount of the mC-compatible probe and the binding amount of the n-mC-compatible probe can be calculated. In this case, additional calculations can be performed. The specific content of the additional calculations is not particularly limited. For example, in addition to arithmetic operations with any number (including the binding amounts of other probes), it can be an exponent of this ratio, a logarithm of this ratio, a power, a root, or a combination thereof. For example, the calculations may be performed independently on the numerator and / or denominator, or on the whole.

[0230] While the determination method based on the bond amount ratio is not limited, for example, a determination value can be calculated using a methylation determination formula, and the determination can be made based on that value.

[0231] The methylation determination formula can be expressed, for example, as follows: (Methylation determination formula) Methylation determination value = Amount of mC-corresponding probe bound / (Amount of mC-corresponding probe bound + Amount of n-mC-corresponding probe bound)

[0232] In the above formula, "mC-compatible probe binding amount" is a measured value indicating the amount of mC-compatible probe bound to the detection region amplification product obtained in the test probe binding step (S0222). In the above formula, "n-mC-compatible probe binding amount" is a measured value indicating the amount of n-mC-compatible probe bound to the detection region amplification product obtained in the test probe binding step (S0222).

[0233] According to this methylation determination formula, a large (close to 1) determination value is obtained if methylation is present, and a small (close to 0) determination value is obtained if methylation is not present.

[0234] By substituting the measured values ​​into the methylation determination formula, a determination value can be calculated. Based on the obtained determination value, it is possible to determine whether or not methylation has occurred at the methylation target site in the detection region on the genomic DNA.

[0235] The determination method based on the aforementioned determination value is not limited, but basically it should be carried out in accordance with the method described in the BS treatment determination step (S0106) above. For example, this could involve setting a cutoff value in advance and determining the presence or absence of methylation of the methylation target site based on that cutoff value, or determining the presence or absence of methylation of the methylation target site based on the statistically significant difference when comparing the determination value obtained in this step with the determination value calculated based on a previously measured methylation standard sample.

[0236] When making a determination based on a cutoff value, a predetermined value is set as the cutoff value. If the determination value is greater than or equal to that value, it can be determined that the methylation target site in the detection region is methylated. Conversely, if the determination value is lower than that value, it can be determined that there is a high probability that the methylation target site is not methylated.

[0237] The method for setting the cutoff value is not particularly limited, but similar to the BS processing determination step (S0106) described above, the determination value group can be classified by percentile based on the relationship between the methylation rate obtained in this step and the determination value, and the percentile value used for that classification can be set as the cutoff value. Specifically, for example, if the 5th percentile of the determination value is set in advance as the cutoff value, and values ​​above that value are defined as methylated and values ​​below that value are defined as unmethylated, then if the determination value calculated by the newly implemented methylation detection method is above the 5th percentile, it can be determined that there is a high probability that the methylation target site in the detection region of the test nucleic acid of that subject is methylated.

[0238] In the case of this process, the method of determination based on "statistical significance" is as follows: When the average determination value calculated based on a standard sample in which the methylation target site on the test nucleic acid is methylated is statistically processed with the determination value of the test subject using the t-test method or the like, if there is no statistically significant difference between the two, it can be determined that there is a high probability that the methylation target site is methylated. On the other hand, if a significant difference is found, it can be determined that there is a high probability that the methylation target site is not methylated. For other matters, the details basically follow those of the BS processing determination process (S0106) described above.

[0239] <Example 1: Verification of the quality of the bisulfite (BS) reaction in the control region of template DNA> (Objective) To verify that in the MLH1 gene promoter methylation detection method of the present invention, the quality of the BS treatment can be determined without being affected by the presence or absence of cytosine methylation in the control region of template DNA.

[0240] (Methods) The control region was defined as the region containing cytosine (nucleotide sequence: CA) that is unaffected by methylation in the β-actin (ACTB) gene. PCR was performed using pre-BS treatment (n-BS treatment) DNA and bisulfite (BS) treated DNA as templates. The amplified products were hybridized onto chips immobilized with various detection probes, and the fluorescence intensity was detected before calculating the result.

[0241] BS treatment was performed using the EZ DNA Methylation-Lightning Kit (ZYMO RESEARCH) according to the attached protocol.

[0242] For the control region amplification primer set, a control region amplification Fw (ACT-F) primer consisting of the nucleotide sequence shown in SEQ ID NO: 8 and a control region amplification Rv (ACT-R) primer consisting of the nucleotide sequence shown in SEQ ID NO: 9 were used. The ACT-R primer was labeled with IC5. PCR amplification was performed according to a standard procedure. PCR cycles consisted of 2 minutes at 94°C, followed by sets of 98°C / 10 seconds, 57°C / 30 seconds, and 68°C / 30 seconds, totaling 40 cycles, with the temperature maintained at 4°C until the end.

[0243] DNA chips, each immobilized with an n-BS detection probe (SEQ ID NO: 11) corresponding to the control region amplification product before BS treatment (n-BS treatment) and a BS detection probe (SEQ ID NO: 10) corresponding to the control region amplification product after BS treatment, were set in a gene analysis instrument (BIOSHOT HT-32; Toyo Kohan Co., Ltd.) and hybridization reactions were performed with the PCR amplification products.

[0244] After the hybridization reaction, the DNA chip needle, to which the DNA chip was immobilized, was immersed in a washing solution tank to wash the DNA chip. After washing, the DNA chip needle was immersed in a detection solution tank, and a 640 nm single-wavelength laser was irradiated, and the excitation light was captured for 2 seconds with a CCD camera.

[0245] The fluorescence intensity values ​​of each measured sample were used to calculate the judgment value by substituting them into the following formula: [Judgment Value] = [Fluorescence Intensity of n-BS Probe] / ([Fluorescence Intensity of BS Probe] + [Fluorescence Intensity of n-BS Probe])

[0246] (Results) The results are shown in Table 1.

[0247]

[0248] In the table, No. 1 shows the judgment value calculated from the fluorescence intensity when the n-mC region before BS treatment (n-BS treatment) is detected using either the n-BS treatment detection probe (n-mC / n-BS detection probe) or the BS treatment detection probe (n-mC / BS detection probe).

[0249] Furthermore, No. 2 shows the judgment value calculated from the fluorescence intensity when the n-mC region after BS treatment is detected using either an n-BS treatment detection probe (n-mC / n-BS detection probe) or a BS treatment detection probe (n-mC / n-BS detection probe).

[0250] From the results above, in sample No. 1 before BS treatment, the judgment value was close to 1, whereas in sample No. 2 after BS treatment, the judgment value was close to 0 regardless of the presence or absence of cytosine methylation. This result suggests that in the MLH1 gene promoter methylation detection method of the present invention, the control region of the template DNA can determine only the quality of the BS treatment without being affected by the presence or absence of cytosine methylation.

[0251] <Example 2: Determination of MLH1 gene promoter methylation using judgment values> (Objective) To confirm that the MLH1 gene promoter methylation detection method of the present invention can determine whether or not the MLH1 gene promoter is methylated in genomic DNA, and to further verify whether it is possible to make a determination according to the percentage of methylation of the promoter.

[0252] (Methods) Using human genomic DNA derived from peripheral blood, and plasmid DNA as a control nucleic acid containing the C and D regions of the human MLH1 gene promoter and the ACTB gene as templates, the mC and n-mC regions contained in each template were adjusted so that the proportion of mC was between 0% and 100%, and then mixed.

[0253] PCR was performed on the detection region and control region of the above template using primer sets capable of amplifying each region, and amplified products were obtained. For amplification of the detection region C, the primer set consisted of an Fw primer with the nucleotide sequence shown in SEQ ID NO: 12 and an Rv primer with the nucleotide sequence shown in SEQ ID NO: 13, which was labeled with IC5. For amplification of the detection region D, the primer set consisted of an Fw primer with the nucleotide sequence shown in SEQ ID NO: 14, which was labeled with IC5, and an Rv primer with the nucleotide sequence shown in SEQ ID NO: 15. Furthermore, for amplification of the control region containing n-mC of the ACTB gene, the primer set consisted of an Fw primer with the nucleotide sequence shown in SEQ ID NO: 8 and an Rv primer with the nucleotide sequence shown in SEQ ID NO: 9, which was labeled with IC5. The PCR conditions were the same as in Example 1.

[0254] Next, DNA chips immobilized with the following probes were placed in a gene analysis device (BIOSHOT HT-32; Toyo Kohan Co., Ltd.): the MLH1-mC probe shown in SEQ ID NO: 4, which corresponds to the mC region amplification product of the C region; the MLH1-n-mC probe shown in SEQ ID NO: 5, which corresponds to the n-mC region amplification product of the C region; the MLH1-mC probe shown in SEQ ID NO: 6, which corresponds to the mC region amplification product of the D region; the MLH1-n-mC probe shown in SEQ ID NO: 7, which corresponds to the n-mC region amplification product of the D region; the BS treatment detection probe shown in SEQ ID NO: 10, which corresponds to the BS treatment control region amplification product; and the n-BS treatment detection probe shown in SEQ ID NO: 11, which corresponds to the non-BS treatment control region amplification product. Hybridization reactions were then performed with the amplification products obtained by PCR.

[0255] After the hybridization reaction, the DNA chip needle, to which the DNA chip was immobilized, was immersed in a washing solution tank to wash the DNA chip. After washing, the DNA chip needle was immersed in a detection solution tank, and a 640 nm single-wavelength laser was irradiated, and the excitation light was captured for 2 seconds with a CCD camera.

[0256] The fluorescence intensity values ​​of each measured sample were introduced into the formula described in Example 1 to calculate the judgment value.

[0257] (Results) Figure 8 shows the results. This figure plots the relationship between the methylation rate in the MLH1 gene promoter and the judgment value. A represents the results for the promoter region C, and B represents the results for the promoter region D.

[0258] In regions C and D, fluorescence intensity and threshold values ​​corresponding to the methylation rate were obtained for both genomic DNA (○) and plasmid DNA (●). The results clearly showed that in both regions, 0% and 5% methylation rates could be clearly separated using a threshold value of 0.1. This suggests that, using the detection method of the present invention, the presence or absence of methylation in a sample can be determined by pre-setting a cutoff value for the threshold value. All publications, patents, and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A kit for determining the accuracy of bisulfite (BS) treatment, comprising: a non-BS treatment detection probe capable of binding to a control region containing one or more unmethylated cytosines in a control nucleic acid before BS treatment; and a BS treatment detection probe capable of binding to the same region in a control nucleic acid after BS treatment.

2. The kit according to claim 1, wherein the control region is a methylated cytosine (mC)-free region.

3. The kit according to claim 1, further comprising a set of control primers for amplifying the control region in a control nucleic acid after BS treatment.

4. The kit according to claim 3, wherein the control primer set is capable of binding to a region that does not contain cytosine.

5. The kit according to any one of claims 1 to 4, further comprising a control nucleic acid.

6. A device for determining the accuracy of BS processing, comprising a non-BS processed detection probe capable of binding to a control region containing cytosine in a control nucleic acid before BS processing, and a BS processed detection probe capable of binding to the same region in a control nucleic acid after BS processing.

7. A method for determining the accuracy of a BS treatment, comprising: a control BS treatment step of performing BS treatment on a control nucleic acid; a control probe binding step of hybridizing a control probe, which is capable of binding to the control region and consists of a BS treatment detection probe and a non-BS treatment detection probe, to the control region after the control BS treatment; a control probe measurement step of measuring the amount of each probe bound after the control probe binding step; and a BS treatment determination step of determining the quality of the BS treatment based on the binding ratio of the BS treatment detection probe and the non-BS treatment detection probe, wherein the control region contains cytosine and the number of mC is known.

8. The method according to claim 7, further comprising a control region amplification step, before the control probe binding step, in which the control nucleic acid after the control BS step is used as a template to amplify the control region by nucleic acid amplification using a control primer set capable of amplifying the control region.

9. The method according to claim 7, wherein the determination is made by determining that the accuracy of the BS treatment was high if the binding ratio calculated using the following formula I is lower than a predetermined cutoff value. Binding ratio = Non-BS treatment detection probe binding amount / (Non-BS treatment detection probe binding amount + BS treatment detection probe binding amount) Formula I 10. A method for detecting methylation in a detection region of a test nucleic acid, comprising: a BS treatment step of BS treatment of a test nucleic acid and a control nucleic acid; a control probe binding step of hybridizing a control probe, comprising a BS-treated detection probe and a non-BS-treated detection probe, which is capable of binding to the control region of the control nucleic acid, to the control region after the BS treatment; a control probe measurement step of measuring the amount of each probe bound after the control probe binding step; a BS treatment determination step of determining the quality of the BS treatment based on the ratio of the amount of BS-treated detection probe bound to the amount of non-BS-treated detection probe bound; a detection region amplification step of amplifying the detection region using the test nucleic acid after the BS treatment step as a template, using a test primer set capable of amplifying the detection region; a test probe binding step of hybridizing an mC-compatible probe and a non-mC-compatible probe to the amplified product after the detection region amplification step; a test amount measurement step of measuring the amount of each probe bound after the test probe binding step; and The method comprising a methylation determination step of determining the presence or absence of methylation in a detection region based on the binding ratio of an mC-compatible probe and a non-mC-compatible probe, wherein the control region contains cytosine, the number of mCs is known, and the methylation determination step is performed only when the BS treatment step is determined to be good.

Images