A methylation detection kit and its application

Abstract

This application provides a methylation detection method, comprising using a modification-sensitive restriction endonuclease, deoxyribonucleoside triphosphates (dNTPs), uracil-DNA glycosyltransferase (UDG), primers that specifically recognize and / or bind to a target sequence, and / or probes that specifically recognize and / or bind to a target sequence. This application also provides a methylation detection kit and detection system prepared based on the above method.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a methylation detection method, a reagent kit or detection system based on the method. Background Technology

[0002] The detection of nucleic acid modifications, particularly DNA methylation, plays a crucial role in the study of gene regulation, disease mechanisms, and other biological processes. DNA methylation, especially at CpG sites, is a key epigenetic modification that can affect gene expression without altering the DNA sequence. Methylation patterns are commonly used as biomarkers in clinical applications, including cancer diagnosis, forensic analysis, and genetic disease research.

[0003] Traditional DNA methylation detection methods typically rely on bisulfite sequencing, methylation-specific PCR (MSP), or microarray techniques. However, these methods have limitations in terms of sensitivity, specificity, and operational complexity. For example, bisulfite treatment can introduce errors, and methylation-specific PCR usually requires high specificity, making the process prone to false positives or false negatives. Furthermore, these methods may require large amounts of starting materials and complex processing steps, thus limiting their practical application in clinical diagnosis or research.

[0004] Recently, methods utilizing methylation-sensitive restriction endonucleases (MSREs) have emerged as a highly efficient and reliable tool for methylation analysis. These enzymes can recognize and cleave specific methylation-sensitive sites, providing a simple and direct approach to detecting methylation status. However, existing detection systems still face challenges in terms of performance, multiplexing capabilities, and ease of operation. Summary of the Invention

[0005] This application provides a nucleic acid detection method, kit, and detection system; specifically, it provides a method, kit, and detection system for detecting nucleic acid methylation status. The advantage of this technical solution lies in combining PCR amplification and enzyme digestion reactions into a single system. This combination makes DNA methylation detection simpler and more efficient, while improving sensitivity and specificity. By fusing the two reactions into one system, or through optimized component ratios and reaction conditions, not only is the experimental procedure simplified, but the throughput and reproducibility of the detection are also improved, making it more widely applicable in practical applications.

[0006] In a first aspect, this application provides a nucleic acid detection kit, the kit comprising one or more selected from the group consisting of: modified sensitive restriction endonucleases, deoxyribonucleoside triphosphates (dNTPs), uracil-DNA glycosylase (UDG), primers or primer pairs capable of specifically recognizing and / or binding to target sequences, and probes capable of specifically recognizing and / or binding to target sequences.

[0007] In some embodiments, the dNTPs include dUTP, dTTP, dATP, dCTP, and dGTP.

[0008] In some embodiments, the ratio of dUTP to dTTP in the dNTP is 1:10 to 10:1, for example, 1:10 to 5:1, 1:10 to 3:1, 1:10 to 1:1, 1:5 to 10:1, 1:3 to 10:1, 1:5 to 5:1, 1:3 to 3:1, 1:2 to 2:1, 1:2 to 1:1, or 1:1 to 2:1. In some embodiments, the ratio of dUTP to dTTP in the dNTP is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1. In some embodiments, the ratio of dTTP to dUTP in the dNTP is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

[0009] In some embodiments, the ratio of dUTP to dTTP is a molar mass ratio. In some embodiments, the ratio of dUTP to dTTP is a mass ratio.

[0010] In some embodiments, the modification-sensitive restriction endonuclease is a methylation-sensitive restriction endonuclease (MSRE).

[0011] In some embodiments, the methylation-sensitive restriction endonuclease is selected from one or more of the following group: BstUI, SmaI, HinP1I, AciI, HpaI, HhaI, HpaII, ApaI, AvaI, and HaeII.

[0012] In some embodiments, the methylation-sensitive restriction endonuclease is selected from one or more of the group consisting of AciI, HpaII, HhaI, and HaeII. In some embodiments, the methylation-sensitive restriction endonuclease includes AciI, HpaII, HhaI, and / or HaeII. In some embodiments, the methylation-sensitive restriction endonuclease is AciI, HpaII, HhaI, and / or HaeII.

[0013] In some embodiments, the methylation-sensitive restriction endonuclease is selected from one or more of the group consisting of: AciI, HhaI, HinP1I, HpaII, AvaI, HaeII, and SmaI. In some embodiments, the methylation-sensitive restriction endonuclease includes AciI, HhaI, HinP1I, HpaII, AvaI, HaeII, and / or SmaI. In some embodiments, the methylation-sensitive restriction endonuclease is AciI, HhaI, HinP1I, HpaII, AvaI, HaeII, and / or SmaI.

[0014] In some embodiments, the concentration of the methylation-sensitive restriction endonuclease is 0.01-0.4 units / μL. For example, the concentration of the methylation-sensitive restriction endonuclease is 0.05-0.4 units / μL, 0.1-0.4 units / μL, 0.2-0.4 units / μL, 0.3-0.4 units / μL, 0.05-0.3 units / μL, or 0.05-0.2 units / μL.

[0015] In some embodiments, the kit further includes nucleic acid polymerase, Mg 2+ Or a buffer solution. In some embodiments, the Mg 2+ Derived from magnesium chloride and / or magnesium sulfate.

[0016] In some embodiments, the buffer is a PCR amplification buffer.

[0017] In some embodiments, the buffer solution is a buffer solution suitable for nucleic acid polymerases (e.g., DNA polymerase) to exert their activity.

[0018] Suitable Mg 2+ This ensures that nucleic acid polymerases (e.g., DNA polymerases) can correctly amplify the target DNA in PCR, or that restriction enzymes can cleave specific sites on the target DNA. In some embodiments, the Mg... 2+ The concentration is 1–10 mM. In some embodiments, the Mg... 2+ The concentration is 1–5 mM, for example 1.5–3 mM. For example, the Mg... 2+ It can come from magnesium chloride or magnesium sulfate.

[0019] In some embodiments, the buffer solution may also contain K + In some implementations, the K + The concentration is 40-60 mM. For example, the K... 2+ It can come from potassium chloride.

[0020] In some embodiments, the pH of the buffer solution is between 7.0 and 9.0. In some embodiments, the pH of the buffer solution is between about 8.0 and about 8.5.

[0021] In some embodiments, the buffer solution may also contain Tris-HCl. For example, it may contain 10-20 mM Tris-HCl. In some embodiments, the buffer solution may also contain DMSO or glycerol.

[0022] In some embodiments, the buffer solution may also contain bovine serum albumin (BSA). For example, the concentration of the BSA may be 1-100 μg / mL.

[0023] In some embodiments, the kit further includes primers or primer pairs that specifically recognize and / or bind to an internal reference gene. In some embodiments, the kit further includes probes that specifically recognize and / or bind to an internal reference gene. In some embodiments, the internal reference gene may be GAPDH, β-actin, or RPL13A.

[0024] Secondly, this application provides a nucleic acid detection system, which includes a modified sensitive restriction endonuclease, deoxyribonucleoside triphosphate (dNTP), uracil-DNA glycosylase (UDG), primers or primer pairs capable of specifically recognizing and / or binding to a target sequence, and a probe capable of specifically recognizing and / or binding to a target sequence.

[0025] In some embodiments, the dNTPs include dUTP, dTTP, dATP, dCTP, and dGTP. In some embodiments, the dNTPs include dUTP, dTTP, dATP, dCT, or dGTP.

[0026] In some embodiments, the ratio of dUTP to dTTP in the dNTP is 1:10 to 10:1, for example, 1:10 to 5:1, 1:10 to 3:1, 1:10 to 1:1, 1:5 to 10:1, 1:3 to 10:1, 1:5 to 5:1, 1:3 to 3:1, 1:2 to 2:1, 1:2 to 1:1, or 1:1 to 2:1. In some embodiments, the ratio of dUTP to dTTP in the dNTP is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1. In some embodiments, the ratio of dTTP to dUTP in the dNTP is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

[0027] In some embodiments, the ratio of dUTP to dTTP is a molar mass ratio. In some embodiments, the ratio of dUTP to dTTP is a mass ratio.

[0028] In some embodiments, the modification-sensitive restriction endonuclease is a methylation-sensitive restriction endonuclease (MSRE).

[0029] In some embodiments, the methylation-sensitive restriction endonuclease is selected from one or more of the following group: BstUI, SmaI, HinP1I, AciI, HpaI, HhaI, HpaII, ApaI, AvaI, and HaeII.

[0030] In some embodiments, the methylation-sensitive restriction endonuclease is selected from one or more of the group consisting of AciI, HpaII, HhaI, and HaeII. In some embodiments, the methylation-sensitive restriction endonuclease includes AciI, HpaII, HhaI, and / or HaeII. In some embodiments, the methylation-sensitive restriction endonuclease is AciI, HpaII, HhaI, and / or HaeII.

[0031] In some embodiments, the methylation-sensitive restriction endonuclease is selected from one or more of the group consisting of: AciI, HhaI, HinP1I, HpaII, AvaI, HaeII, and SmaI. In some embodiments, the methylation-sensitive restriction endonuclease includes AciI, HhaI, HinP1I, HpaII, AvaI, HaeII, and / or SmaI. In some embodiments, the methylation-sensitive restriction endonuclease is AciI, HhaI, HinP1I, HpaII, AvaI, HaeII, and / or SmaI.

[0032] In some embodiments, the concentration of the methylation-sensitive restriction endonuclease is 0.01-0.4 units / μL. For example, the concentration of the methylation-sensitive restriction endonuclease is 0.05-0.4 units / μL, 0.1-0.4 units / μL, 0.2-0.4 units / μL, 0.3-0.4 units / μL, 0.05-0.3 units / μL, or 0.05-0.2 units / μL.

[0033] In some embodiments, the nucleic acid detection system further includes nucleic acid polymerase and Mg2+. 2+ Or a buffer solution. In some embodiments, the Mg 2+ Derived from magnesium chloride and / or magnesium sulfate.

[0034] In some embodiments, the buffer is a PCR amplification buffer.

[0035] In some embodiments, the buffer solution is a buffer solution suitable for nucleic acid polymerases (e.g., DNA polymerase) to exert their activity.

[0036] Suitable Mg 2+ This ensures that nucleic acid polymerases (e.g., DNA polymerases) can correctly amplify the target DNA in PCR, or that restriction enzymes can cleave specific sites on the target DNA. In some embodiments, the Mg... 2+ The concentration is 1–10 mM. In some embodiments, the Mg... 2+ The concentration is 1–5 mM, for example 1.5–3 mM. For example, the Mg... 2+ It can come from magnesium chloride or magnesium sulfate.

[0037] In some embodiments, the buffer solution may also contain K + In some implementations, the K + The concentration is 40-60 mM. For example, the K... 2+ It can come from potassium chloride.

[0038] In some embodiments, the pH of the buffer solution is between 7.0 and 9.0. In some embodiments, the pH of the buffer solution is between about 8.0 and about 8.5.

[0039] In some embodiments, the buffer solution may also contain Tris-HCl. For example, it may contain 10-20 mM Tris-HCl. In some embodiments, the buffer solution may also contain DMSO or glycerol.

[0040] In some embodiments, the buffer solution may also contain bovine serum albumin (BSA). For example, the concentration of the BSA may be 1-100 μg / mL.

[0041] In some embodiments, the nucleic acid detection system further includes primers or primer pairs that specifically recognize and / or bind to an internal reference gene. In some embodiments, the nucleic acid detection system further includes probes that specifically recognize and / or bind to an internal reference gene. In some embodiments, the internal reference gene may be GAPDH, β-actin, or RPL13A.

[0042] Thirdly, this application provides a nucleic acid detection method (e.g., a methylation detection method), the method comprising: (a) mixing a nucleic acid sample with the methylation detection system described in this application to obtain a mixture; (b) maintaining the mixture for 15-120 min under conditions that allow the modification-sensitive restriction endonuclease to exert its enzymatic digestion activity to obtain an enzyme digestion product; and (c) amplifying the enzyme digestion product obtained in step (b).

[0043] In some embodiments, (b) includes maintaining the mixture under conditions that allow the modified-sensitive restriction endonuclease to exert its cleavage activity for 25-120 min, 30-120 min, 45-120 min, 60-120 min, 80-120 min, 90-120 min, 100-120 min, 15-110 min, 15-100 min, 15-90 min, 15-80 min, 15-70 min, 15-60 min, or 15-50 min, for example, for 30-120 min.

[0044] In some embodiments, the conditions under which the modification-sensitive restriction endonuclease exerts its digestive activity include room temperature. In some embodiments, the conditions under which the modification-sensitive restriction endonuclease exerts its digestive activity include a temperature of 37°C. In some embodiments, the conditions under which the modification-sensitive restriction endonuclease exerts its digestive activity include a temperature of 30°C.

[0045] In some embodiments, the amplification includes heating the mixture to above 85°C and holding it for at least 5 minutes. In some embodiments, the amplification includes heating the mixture to above 90°C and holding it for at least 5 minutes. In some embodiments, the amplification includes heating the mixture to above 95°C and holding it for at least 5 minutes. In some embodiments, the amplification includes heating the mixture to above 85°C and holding it for at least 10 minutes. In some embodiments, the amplification includes heating the mixture to above 85°C and holding it for at least 15 minutes. In some embodiments, the amplification includes heating the mixture to above 90°C and holding it for at least 10 minutes.

[0046] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0047] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0048] Figure 1 The display shows the amplification curves detected using this detection method, with gradient methylation standards having expected methylation rates of 100%, 50%, 25%, 10%, 5%, 1%, or 0%.

[0049] Figure 2 The figure shows the consistency between the measured methylation rate and the expected methylation rate using the method of this application.

[0050] Figure 3 The figure shows the measured methylation rate of 100% methylation standard at various enzyme digestion times using the method of this application.

[0051] Figure 4 The figure shows the measured methylation rate of 50% methylated standard at various enzyme digestion times using the method of this application.

[0052] Figure 5 The figure shows the measured methylation rate at various enzyme digestion times for 0% methylated standard detected using the method of this application.

[0053] Figure 6 The displayed curve is the MS-34 standard curve of self-prepared reaction buffer 1.

[0054] Figure 7 The displayed curve is the MS-34 standard curve of self-prepared reaction buffer 2.

[0055] Figure 8 The displayed curve is the MS-34 standard curve of self-prepared reaction buffer 3.

[0056] Figure 9 The displayed curve is the MS-34 standard curve of the original PCR reaction buffer (control group).

[0057] Figure 10 The display shows the measured average methylation rate distribution of the 100% methylated reference material.

[0058] Figure 11 The display shows the measured average methylation rate distribution of the 50% methylated reference material.

[0059] Figure 12 The display shows the measured average methylation rate distribution of the 0% methylated reference material. Detailed Implementation

[0060] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0061] Terminology Definition

[0062] In this application, the term "deoxyribonucleoside triphosphate" (dNTP) generally refers to the triphosphate form of a nucleotide used in DNA synthesis. dNTPs may include dATP (deoxyadenosine triphosphate), dTTP (deoxythymidine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate), and dUTP (deoxyuridine triphosphate). During DNA synthesis, dUTPs can substitute for dTTPs in the synthetic reaction.

[0063] In this application, the term "endonuclease" refers to an enzyme that cuts at a specific site within a nucleic acid molecule. The terms "endonuclease" and "restriction endonuclease" are used interchangeably. The term "methylation-sensitive endonuclease (MSE)" is used interchangeably with "methylation-sensitive restriction endonuclease (MSRE)." In this application, the terms "methylation-sensitive restriction endonuclease" or "MSRE" refer to an endonuclease whose activity increases or decreases when one or more methylation sites are present in the DNA sequence. Thus, in one embodiment, the activity of MSRE is reduced or inhibited when DNA is methylated. In another embodiment, the activity of MSRE increases when DNA is methylated (e.g., methylation-dependent endonucleases). More information about methylation-sensitive restriction endonucleases can be found in the Restriction Endonuclease REBASE database: http: / / rebase.neb.com / rebase / .

[0064] In this application, the term "buffer solution" generally refers to a solution used to maintain the specific pH and ionic strength required for nucleic acid amplification or enzymatic reactions. This buffer solution typically contains appropriate amounts of buffering components (such as Tris-HCl, HEPES, or PBS), salts (such as KCl, NaCl), and magnesium ions (Mg²⁺). 2+ ), or other auxiliary ions, are added to provide a suitable reaction environment, ensuring optimal enzyme activity and reaction stability. Furthermore, the composition of the buffer can be adjusted according to specific applications, such as PCR amplification buffer or restriction enzyme digestion buffer.

[0065] In this application, the term "internal reference gene" generally refers to a gene used as a reference in experiments whose expression level remains relatively stable under different experimental conditions. Internal reference genes are typically used to correct for differences between samples, such as differences in nucleic acid extraction volume or reaction efficiency, thereby helping to accurately assess the expression level of target genes. Common internal reference genes include, but are not limited to, GAPDH, β-actin, and 18S rRNA. The expression levels of these genes remain constant in most cell types and under most conditions, and therefore they are often used as a standardized reference in quantitative PCR (qPCR) and other gene expression analysis experiments.

[0066] In this application, the term "probe" generally refers to a short-chain nucleic acid molecule used to specifically recognize and bind to a target nucleic acid sequence. Probes achieve recognition through complementary pairing with the target sequence and are commonly used in molecular detection techniques such as quantitative real-time PCR (qPCR), in situ hybridization, or gene chip analysis. Probe design typically considers its specific binding ability to the target sequence and whether it contains a tag that can be used for detection, such as a fluorescent label, radioisotope, or chemiluminescent label.

[0067] In this application, the term "primer" generally refers to a short-chain nucleic acid molecule, typically composed of about 18-30 nucleotides, used to initiate the synthesis of DNA or RNA in nucleic acid amplification reactions (such as PCR or LAMP). Primers initiate the synthesis reaction of DNA polymerase through complementary pairing with a target sequence. Primers can be classified into forward primers (used to extend the 5' end of the target sequence) and reverse primers (used to extend the 3' end of the target sequence). Invention Details

[0069] An illustrative and non-limiting scheme for methylation analysis according to this application is illustrated below.

[0070] 1. Detection Method

[0071] This application provides a method for detecting methylation of DNA sequences in a sample, wherein enzyme digestion and amplification are performed in the same system, and an UDG anti-contamination system is added. This application also provides a kit and detection system for the above method.

[0072] In this application, the enzyme digestion system may include dNTPs and MSRE, the amplification system may include primers, probes, and nucleic acid polymerase, and the contamination prevention system may use uracil-DNA glycosylation enzyme.

[0073] In one embodiment, the sample to be tested is contacted with an MSRE digestion and qPCR amplification reaction mixture. In the reaction mixture, MSRE, DNA polymerase (e.g., thermophilic DNA polymerase or a hot-start DNA polymerase system), oligonucleotide primers, dNTPs, and buffer are formulated to promote the activity of both the MSRE and the DNA polymerase. The reaction mixture may also contain a DNA-binding label for detecting the presence of DNA. The salt concentration in the reaction mixture may vary depending on the MRSE and polymerase used.

[0074] The MSRE used in the methods of this application can be a naturally occurring or engineered enzyme. For example, the MSRE can be an enzyme that blocks DNA methylation, such as AatII, AccIII, Acil, AfaI, Agel, AhaII, Alw26I, Alw44I, ApaLI, ApyI, AscI, Asp718I, AvaI, AvaII, Bme216I, AvaI, AvaII, Bme216I, BsaAI, BsaHI, BscFI, BsiMI, BsmAI, BsiEI, BsiWI, BsoFI, Bsp105I, Bsp119I, BspDI, BspEI, BspHI, BspKT6I, BspMII, BspRI, BspT104I, BsrFI, BssHII, BstBI, BstEIII, BstUI, BsuFI, BsuRI, Cad, CboI, Cb rI, CceI, Cfr10I, ClaI, Csp68KII, Csp45I, CtyI, CviAI, CviSIII, DpnII, EagI, Ecl136II, Eco47I, Eco47III, EcoRII, EcoT22I, EheI, Esp3I, Fnu4HI, FseI, FspI, Fsp4HI, GsaI, HaeII , HaeIII, HgaI, HhaI, HinP1I, HpaII, HpyAIII, ItaI, KasI, Kpn2I, LlaAI, LlaKR2I, MboI, MflI, MluI, MmeII, MroI, MspI, MstII, MthTI, NaeI, NarI, NciAI, NdeII, NgoMIV, NgoPII, NgoS II, NlaIII, NlaIV, NotI, NruI, NspV, PmeI, PmlI, Psp1406I, PvuI, RalF40I, RsaI, RspXI, RsrII, SacII, SalI, Sau3AI, SexAI, SfoI, SfuI, SmaI, SnaBI, SolI, SpoI, SspRFI, Sth368I, Tail, TaqI, TflI, TthHB8I, VpaK11BI, or XhoI. In one embodiment, the MSRE can be an enzyme that cleaves DNA only in the presence of methylation, such as AciI, HpaII, HhaI, and HaeII. In one embodiment, the MSRE can be AciI, HhaI, HinP1I, HpaII, AvaI, HaeII, and SmaI.

[0075] Samples for testing can be obtained from various sources, typically DNA samples. In some cases, multiple DNA samples are analyzed in parallel. Samples used for parallel analysis may include DNA standards with known methylation levels. Similarly, a variety of different oligonucleotide probes can be used to determine the methylation status of multiple regions in a DNA sample.

[0076] The method of this application may include incubating a sample under conditions where MSRE can cut DNA. The buffer and temperature conditions for MSRE cutting may be adjusted according to the specific MSRE enzyme used and further details described herein. For example, incubation at 37°C may be used with AciI, HpaII, HhaI, or HaeII.

[0077] The method described in this application may include incubating a sample under conditions suitable for DNA polymerization. qPCR methods and reagents are well known in the art and can be modified based on the description herein to achieve quantitative amplification of DNA samples. In some embodiments, the sample is incubated in a thermal cycler for 25-45 rounds of amplification, the temperature used facilitating primer denaturation, primer annealing with sample DNA, and extension of intact sample DNA.

[0078] The method described in this application may include detecting DNA amplification. qPCR methods for detecting DNA amplification are known in the art and are exemplified herein. For example, fluorescent dyes binding double-stranded DNA can be detected. In this case, increased fluorescence indicates DNA amplification. Similarly, labeled DNA probes can also be used to detect amplification. In some aspects, multiple tags or dyes may be included in a single reaction.

[0079] The method of this application may include determining the methylation ratio in a DNA sample. The DNA amplification detected above can be used to determine the Ct (including inactive MSRE) for each test and control reaction.

[0080] 2. Sample

[0081] Samples usable in this application may be eukaryotic genomic DNA, including but not limited to mammalian DNA such as rodents, mice, rats, rabbits, guinea pigs, even-toed ungulates, horses, sheep, pigs, goats, cattle, cats, dogs, primates, humans, or non-human primates. Plant DNA can also be analyzed using the methods of this application. For example, DNA from araffinose, maize, sorghum, oats, wheat, rice, rapeseed, or soybeans can be analyzed. Genomic DNA from other organisms can also be analyzed, such as algae, nematodes, insects (fruit flies, mosquitoes, bees, or spiders), fish, reptiles, amphibians, and yeast.

[0082] Genomic DNA can be isolated from one or more cells, bodily fluids, or tissues. A variety of methods can be used to isolate genomic DNA from samples such as blood, sweat, tears, lymph, urine, saliva, semen, cerebrospinal fluid, feces, or amniotic fluid. Genomic DNA can also be obtained from one or more cells or tissues, which can be primary cultured cells, propagated cell lines, fixed archival samples, forensic samples, or archaeological samples. Methods for isolating genomic DNA are well known in the technical field.

[0083] Typical cell types from which genomic DNA can be obtained in the method of this application include: blood cells, such as B lymphocytes, T lymphocytes, leukocytes, erythrocytes, macrophages, or neutrophils; muscle cells, such as skeletal muscle cells, smooth muscle cells, or cardiomyocytes; germ cells, such as sperm or eggs; epithelial cells; connective tissue cells, such as adipocytes, fibroblasts, or osteoblasts; neurons; astrocytes; stromal cells; kidney cells; pancreatic cells; hepatocytes; or keratinocytes. The cells from which genomic DNA is obtained can be at specific developmental stages, including, for example, hematopoietic stem cells or cells derived from hematopoietic stem cells, such as erythrocytes, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, macrophages, or platelets. Generally, any type of stem cell can be used, including but not limited to embryonic stem cells, adult stem cells, totipotent stem cells, or pluripotent stem cells.

[0084] The cells from which genomic DNA samples are obtained can be normal cells or cells exhibiting symptoms of certain diseases or conditions. The genomic DNA used in the methods of this application can be derived from cancer cells, tumor cells, apoptotic cells, senescent cells, necrotic cells, autoimmune cells, cells carrying hereditary diseases, etc.

[0085] The DNA used in the methods of this application can be a standard or reference DNA sample. These reference samples may contain known levels of DNA methylation. For example, a reference DNA sample may be DNA extracted from cells lacking one or more DNA methyltransferases and with almost no CpG methylation. Alternatively, a reference DNA sample may be treated with a DNA methyltransferase (such as M. SsssI methyltransferase) and thus contain methylation at almost all methylable CpG sites. For example, the standard DNA may be DNA extracted from human cell lines, such as healthy human leukocytes. In some embodiments, the methods of this application involve using two or more standard DNA samples, such as DNA samples with almost no CpG methylation, DNA samples with a known specific level of CpG methylation, and DNA samples with almost complete CpG methylation.

[0086] 3. Reagents and Kits

[0087] The kit may contain all the components required for efficient MSRE DNA digestion and PCR. In some embodiments, the kit may contain a premix. For example, the premix may contain one, two, three, four or more MSREs, DNA polymerases, nucleotides, and a salt / buffer system designed to maximize the activity of the polymerases and MSEs. The premix may also contain tags that can be used to quantify PCR amplification in a sample. For example, the tags may be fluorescent tags that bind to amplified double-stranded DNA, or nucleic acid probes that specifically bind to amplified DNA. In some aspects, the premix may also contain oligonucleotide primers. In some embodiments, these primers may also be packaged separately. In some aspects, the premix may also contain an anticontamination system. Reagents containing anticontamination components, such as the addition of DNA removal enzymes (e.g., uracil-DNA glycosylase), can be used to eliminate exogenous DNA contamination, as these enzymes degrade contaminating DNA in the PCR reaction system.

[0088] The kit may also include, but is not limited to, oligonucleotide primers, reference DNA samples (such as methylated and unmethylated reference samples), sterile water, probes, dyes, sample vials, and instructions describing the detection method steps. In some further aspects, the kit may also include reagents for DNA isolation, DNA purification, and / or DNA cleaning.

[0089] The reagent kit components can be packaged in aqueous solutions or lyophilized forms. The kit container typically includes at least one vial, test tube, or other container into which the reagent can be placed, preferably appropriately dispensed. If the kit contains multiple components, it usually also includes second, third, or other additional containers into which the additional components can be placed separately. However, various combinations of components can also be packaged in a single vial. When the kit components are provided as one or more liquid solutions, the liquid solution is an aqueous solution, preferably a sterile aqueous solution. The kit components can also be provided as dry powders. When reagents and / or components are provided as dry powders, the powder can be reconstituted by adding a suitable solvent.

[0090] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the reagent kits, detection methods, etc. of this application, and are not intended to limit the scope of the invention.

[0091] Example

[0092] Example 1: MSRE-qPCR enzyme digestion and amplification to detect sample methylation

[0093] 1.1 Preparation of anti-pollution dNTP mix

[0094] Taking 100 PCR reactions as an example, we used 100mM dATP, dTTP, dGTP, dCTP, and dUTP solutions from Novizan and nuclease-free water from Ambion. Following the dosages in Table 1, we added each solution sequentially to 5mL centrifuge tubes. Vortex for 2 minutes to ensure thorough mixing, resulting in a contamination-free dNTP mix. Aliquot and store at -20℃ for later use.

[0095] Table 1 dNTP mix formulation

[0096]

[0097]

[0098] 1.2 Preparation of Enzyme Digestion Amplification Reaction Solution

[0099] Taking two PCR reactions as an example, the following were used: Novizan's Taq HSDNA Polymerase, PCR Enhancer, and Heat-labile UDG; TransGen's Passive Reference Dye II (50×); Ambion's nuclease-free water; and the contamination-resistant dNTP mix prepared in step 1.1. According to Table 2, these were added sequentially to 1.5 mL centrifuge tubes. The tubes were vortexed for 2 min to ensure thorough mixing, yielding the enzyme digestion and amplification reaction solution, which was then stored at -20°C or kept for later use.

[0100] Table 2. Enzyme digestion and amplification reaction solution formulation

[0101] Reagent Name Usage amount (μL) 10×Taq HS Buffer (Mg2+ plus) 5 Taq HSDNA polymerase (5 U / μL) 1.3 dUTP / dUTP Mix 1.5 PCR Enhancer 10 Heat-labile UDG (1 U / μL) 1 Passive Reference Dye II(50×) 0.5 Nuclease-free water 0.7 Total volume 20

[0102] 1.3 Preparation of primer and probe working solutions

[0103] First, upstream and downstream primers and fluorescently labeled probe oligonucleotide chains for target gene amplification were synthesized by Sangon Biotech and provided in 100 μM solution form. NanoDrop was then used. TM The concentration of the primer and probe solution provided by the supplier was determined using a One / OneC micro-UV-Vis spectrophotometer in ssDNA mode. The actual concentration was calculated, and the primer and probe were diluted to a final concentration of 20 μM using nuclease-free water.

[0104] Taking 10 PCR reactions as an example, according to Table 3, add the 20 μM solution of each primer and probe to a 1.5 mL centrifuge tube in sequence. Vortex for 2 min to thoroughly mix the components and obtain the primer and probe working solution, which can be stored at -20℃ or used for later use.

[0105] Table 3 Primer and probe working solution formulations

[0106]

[0107]

[0108] 1.4 Construction of the enzyme digestion and amplification reaction system

[0109] Taking one enzyme digestion and amplification reaction system as an example, the following were used: the anti-contamination dNTP mix, enzyme digestion and amplification reaction solution, primer and probe working solution prepared in steps 1.1-1.3, and methylation-sensitive restriction endonucleases (MSRE) AciI, HpaII, HhaI, and HaeII purchased from NEB. According to Table 4, these were added sequentially to 1.5 mL centrifuge tubes. After vortexing for 2 min to ensure thorough mixing, the mixture was aliquoted into MicroAmp at 14 μL / tube. TM It is kept in the optical 8-tube array for later use.

[0110] Table 4. Formulation of the enzyme digestion and amplification reaction system

[0111] Reagent Name Usage amount (μL) Enzyme digestion and amplification reaction solution 10 Primer and probe working solution (20 μM) 2.5 AciI 0.5 HpaII 0.5 HhaI 0.25 HaeII 0.25 Total volume 14

[0112] When using the prepared enzyme digestion and amplification reaction system, add 11 μL of DNA sample, vortex to mix, and then centrifuge briefly before placing it in a qPCR instrument and running the program in Table 5 for detection.

[0113] Table 5 MSRE-qPCR reaction conditions

[0114]

[0115] Experimental results: The fully methylated standard produced by Takara Methylated HCT116g DNA was mixed with DNA from leukocytes that were confirmed negative by methylation capture sequencing at ratios of 100%, 50%, 25%, 10%, 5%, 1%, and 0% (percentage of fully methylated standard, ng / ng) to prepare gradient methylation standards. The expected methylation rates were 100%, 50%, 25%, 10%, 5%, 1%, and 0%, respectively. 15ng of each gradient standard was used for detection using this method, and the amplification curves are shown below. Figure 1 As shown in Table 6. CT and dCT are also shown. A comparison of the consistency between the measured methylation rate and the expected methylation rate calculated based on ddCT is shown below. Figure 2 As shown in the figure. This method can distinguish templates with different methylation rates and provides accurate quantitative results.

[0116] Table 6. Methylation Standards CT and dCT at Different Gradients

[0117] Expected methylation rate Target name Cт Target name Cт dCT 100％ MS-50 26.99 U-ACTB 24.91 2.08 100％ MS-50 26.98 U-ACTB 24.88 2.10 50％ MS-50 28.10 U-ACTB 25.05 3.06 50％ MS-50 28.01 U-ACTB 25.06 2.95 25％ MS-50 29.00 U-ACTB 25.13 3.87 25％ MS-50 29.02 U-ACTB 25.30 3.72 10％ MS-50 30.65 U-ACTB 25.43 5.22 10％ MS-50 30.57 U-ACTB 25.35 5.22 5％ MS-50 31.61 U-ACTB 25.31 6.31 5％ MS-50 31.64 U-ACTB 25.48 6.16 1％ MS-50 33.56 U-ACTB 25.39 8.17 1％ MS-50 33.40 U-ACTB 25.52 7.88 0％ MS-50 37.40 U-ACTB 25.32 12.07 0％ MS-50 36.62 U-ACTB 25.46 11.16

[0118] Example 2: Detection results of different enzyme digestion times

[0119] Following the method in Example 1, the detection effect on sample methylation at different enzyme digestion times was examined. A gradient of methylation standards with expected methylation rates of 100%, 50%, and 0% was used, with enzyme digestion times of 0 min, 15 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, 120 min, 135 min, 150 min, and 165 min. The results are as follows: Figures 3-5 The results are shown in Tables 7-9. The results indicate that the detection results for standards with different methylation levels within the enzyme digestion time range of 30 to 120 minutes were consistent with expectations.

[0120] Table 7. Measured methylation rate of 100% methylation standard at various enzyme digestion times.

[0121]

[0122]

[0123] Table 8. Measured methylation rate of 50% methylation standard at various enzyme digestion times.

[0124] methylation rate Enzyme digestion time Measured average methylation rate Standard deviation 50％ 0min 99.3％ 0.05 50％ 15min 81.0％ 0.05 50％ 30min 48.4％ 0.02 50％ 45min 48.4％ 0.02 50％ 60min 49.5％ 0.00 50％ 75min 49.2％ 0.02 50％ 90min 49.5％ 0.00 50％ 105min 49.6％ 0.01 50％ 120min 48.4％ 0.01 50％ 135min 40.7％ 0.02 50％ 150min 40.1％ 0.02 50％ 165min 37.8％ 0.03

[0125] Table 9. Measured methylation rate of 0% methylation standard at various enzyme digestion times.

[0126] methylation rate Enzyme digestion time Measured average methylation rate Standard deviation 0％ 0min 99.5％ 0.01 0％ 15min 29.7％ 0.00 0％ 30min 0.0％ 0.00 0％ 45min 0.0％ 0.00 0％ 60min 0.1％ 0.00 0％ 75min 0.0％ 0.00 0％ 90min 0.0％ 0.00 0％ 105min 0.0％ 0.00 0％ 120min 0.0％ 0.00 0％ 135min 0.0％ 0.00 0％ 150min 0.0％ 0.00 0％ 165min 0.0％ 0.00

[0127] Example 3: Study on the anti-contamination effect of reaction buffer on UDG enzyme

[0128] Using MS-34 target (hg19 chr17:35300789-35300942) as the research object, the amplification products of the MS-34 anti-contamination reaction system were diluted to 1000 copies of contaminant test samples per PCR reaction. These samples were digested in UNG restriction enzyme digestion systems containing three self-prepared reaction buffers and a manufacturer's recommended reaction buffer (control), with six technical replicates for each reaction buffer. The digestion products were analyzed using quantitative real-time PCR to detect the CT value of the MS-34 contamination template. Self-prepared reaction buffers showing no amplification signal (CT = NoCt) in each technical replicate could be used to construct a one-step MSRE-qPCR reaction system. The three self-prepared reaction solutions were composed of the following components: 10-20 mM Tris-HCl (pH 7-8), 40-60 mM potassium chloride, 1-10 mM magnesium chloride or magnesium sulfate, and 1-100 μg / mL bovine serum albumin (BSA). The specific implementation scheme is as follows:

[0129] 1. Test Sample Preparation

[0130] The amplification product from the MS-34 anti-contamination reaction system was collected. After concentration detection using Qubit HS quantitative reagent, it was serially diluted to the theoretical concentration of 3.4 × 10⁻⁶. -7 ng / μL. Add nuclease-free (NF)-free water to a total volume of 528 μL (enough for 24 reactions) to obtain 1000 copies of contaminant / reaction test sample;

[0131] 2. UDG enzyme digestion

[0132] Prepare the UDG digestion reaction system in an ice box according to Table 10 and add the sample to be tested. Set up the PCR instrument according to Table 11. Place the reaction system in the PCR instrument and run the digestion program. After the reaction is complete, purify the reaction product using 1.8× magnetic beads, wash off the magnetic beads with 10 μL of NF water to obtain the qPCR template;

[0133] Table 10 Anti-pollution enzymatic digestion reaction system

[0134] Components Volume (μL) 10× reaction buffer 2.5 UDG enzyme (1u / μL) 0.5 DNA 22 Total volume 25μL

[0135] Table 11 Enzyme digestion reaction conditions

[0136] temperature time 37℃ 60min 95℃ 2min

[0137] 3. qPCR detection

[0138] Prepare the qPCR reaction system according to Table 12. Place the reaction system on the heating module of the qPCR instrument. After confirming that the tube caps are sealed, close the sample chamber door. Set the fluorescence channel for each detection tube according to Table 13, and set the qPCR amplification program according to Table 14, and then start the detection.

[0139] Table 12 qPCR reaction system

[0140] Components Volume (μL) PCR reaction solution 10 MS-34 primer-probe mixture 5 DNA 10 Total volume 25

[0141] Table 13 Fluorescence Channel Settings

[0142] Gene signal channel MS-34 FAM

[0143] Table 14 Amplification Procedure

[0144]

[0145] 4. Data Analysis

[0146] The MS-34 detection results are shown in Table 15. No MS-34 amplification signal was detected in any of the three self-prepared reaction buffers or the control reaction buffer. This indicates that the UDG enzyme exhibits normal digestion activity in both the three self-prepared reaction buffers and the manufacturer's original reaction buffer, with no MS-34 residue, achieving a contamination prevention effect. Its performance meets the contamination prevention requirements of qPCR and can be used for the construction of a one-step MSRE-qPCR amplification system. One-step MSRE-qPCR means that the methylation-sensitive restriction enzyme and PCR reaction are carried out in one system or container without separation.

[0147] Table 15 Measured data for each reaction buffer solution

[0148] reaction well Sample Name Target name Cт A1 Buffer1-NTC-1 MS-34F1 / R1 NoCt B1 Buffer1-NTC-2 MS-34F1 / R1 NoCt C1 Buffer1-NTC-3 MS-34F1 / R1 NoCt D1 Buffer1-NTC-4 MS-34F1 / R1 NoCt E1 Buffer1-NTC-5 MS-34F1 / R1 NoCt F1 Buffer1-NTC-6 MS-34F1 / R1 NoCt A2 Buffer2-NTC-1 MS-34F1 / R1 NoCt B2 Buffer2-NTC-2 MS-34F1 / R1 NoCt C2 Buffer2-NTC-3 MS-34F1 / R1 NoCt D2 Buffer2-NTC-4 MS-34F1 / R1 NoCt E2 Buffer2-NTC-5 MS-34F1 / R1 NoCt F2 Buffer2-NTC-6 MS-34F1 / R1 NoCt A3 Buffer3-NTC-1 MS-34F1 / R1 NoCt B3 Buffer3-NTC-2 MS-34F1 / R1 NoCt C3 Buffer3-NTC-3 MS-34F1 / R1 NoCt D3 Buffer3-NTC-4 MS-34F1 / R1 NoCt E3 Buffer3-NTC-5 MS-34F1 / R1 NoCt F3 Buffer3-NTC-6 MS-34F1 / R1 NoCt A4 Control-NTC-1 MS-34F1 / R1 NoCt B4 Control-NTC-2 MS-34F1 / R1 NoCt C4 Control-NTC-3 MS-34F1 / R1 NoCt D4 Control-NTC-4 MS-34F1 / R1 NoCt E4 Control-NTC-5 MS-34F1 / R1 NoCt F4 Control-NTC-6 MS-34F1 / R1 NoCt

[0149] Example 4: Study on the amplification efficiency of self-prepared reaction buffer for qPCR

[0150] Using MS-34 as the target, plasmid standards containing the target fragment were detected in three self-prepared reaction buffers (described in Example 3) and the manufacturer's qPCR reagent reaction buffer (used for primer and probe quality control). The plasmid standard amounts were 10^6, 10^5, 10^4, 10^3, 100, 50, and 25 copies, with three technical replicates for each copy number gradient. The self-prepared reaction buffer with the amplification efficiency closest to 100% was used for constructing a one-step MSRE-qPCR reaction system. The specific implementation scheme is as follows:

[0151] 1. Test Sample Preparation

[0152] Take the artificially synthesized plasmid standard carrying the MS-34 target sequence and dilute it in a gradient of 10^6, 10^5, 10^4, 10^3, 100, 50, and 25 copies / 10 μL according to Table 16.

[0153] Table 16 Dilution Dosage of MS-34 Plasmid Standard

[0154]

[0155] 2. qPCR detection

[0156] Prepare the qPCR reaction system according to Table 17. Place the reaction system on the heating module of the qPCR instrument, and after confirming that the tube caps are sealed, close the sample chamber door. Set the fluorescence channel for each detection tube according to Table 18, and set the qPCR amplification program according to Table 19, then start the detection.

[0157] Table 17 qPCR reaction system

[0158] Components Volume (μL) PCR reaction solution 10 MS-34 primer-probe mixture 5 plasmid standard 10 Total volume 25

[0159] Table 18 Fluorescence Channel Settings

[0160] Gene signal channel MS-34 FAM

[0161] Table 19 Amplification Procedure

[0162]

[0163] 3. Data Analysis

[0164] The MS-34 test results are shown in Table 20, and the standard curve is shown in Table 20. Figure 6-9 The amplification efficiencies are shown in Table 21. The results show that the amplification efficiency of all buffers was close to 100%. This indicates that the performance of the self-prepared reaction buffers is basically the same as the control buffers, and both meet the requirements of qPCR for primer and probe amplification efficiency. Therefore, all three self-prepared buffers can be used to construct a one-step MSRE-qPCR amplification system.

[0165] Table 20: Standard curve detection data for each reaction buffer.

[0166]

[0167]

[0168] Table 21 MS-34 amplification efficiency in each reaction buffer.

[0169] buffer solution slope efficiency <![CDATA[R 2 <!-- 17 -->]]> Self-prepared reaction buffer 1 -3.3303 99.7％ 0.997 Self-prepared reaction buffer 2 -3.3296 99.7％ 0.9985 Self-prepared reaction buffer 3 -3.3167 100.2％ 0.9989 Original manufacturer's PCR reaction buffer (control) -3.3237 99.9％ 0.9991

[0170] Example 5: Study on digestion time of methylation-sensitive restriction endonuclease

[0171] Using MS-34 as the target, and employing a self-prepared reaction buffer from Example 4, the digestion times of seven methylation-sensitive restriction endonucleases (AciI, HhaI, HinP1I, HpaII, AvaI, HaeII, and SmaI) involved in the target technical scheme were determined using gradient methylation rate references (methylation rates of 100%, 50%, and 0%). By comparing the measured methylation rate with the expected methylation rate, the time period during which the measured methylation rate and the expected methylation rate were consistent was determined as the optimal digestion reaction time. The specific implementation scheme is as follows:

[0172] For reaction system preparation, prepare the enzyme digestion reaction system in an ice box according to Table 22. The 0-minute enzyme digestion reaction system needs to be prepared before qPCR.

[0173] Table 22 Enzyme Digestion PCR Reaction System

[0174] Components Volume (μL) PCR reaction solution 10 MS-34 primer-probe mixture 5 7 MSRE enzyme digestion mixtures 5.5 DNA 4.5 (15ng) Total volume 25

[0175] Samples digested for 165 minutes were placed in a standard PCR instrument, and the reaction program in Table 23 was run. Then, following the order of digestion time from highest to lowest, samples were placed in the PCR instrument every 15 minutes. After digestion, the samples were briefly centrifuged and then transferred to a qPCR instrument to determine the methylation rate.

[0176] Table 23 Enzyme digestion reaction conditions

[0177] stage temperature time 1 37℃ 165min 2 95℃ 10min

[0178] Transfer the enzyme digestion products and non-enzyme digestion reaction system to the heating module of the qPCR instrument. After confirming that the tube caps are sealed again, close the sample chamber door. Set the fluorescence channel for each detection tube according to Table 24, set the qPCR amplification program according to Table 25, and then start the detection.

[0179] Table 24 Fluorescence Channel Settings

[0180] Gene signal channel MS-34 FAM

[0181] Run the program in Table 25 to detect the methylation rate of each reference sample at different enzyme digestion times.

[0182] Table 25 qPCR Detection Reaction Conditions

[0183]

[0184] After exporting the detection data, the dCT value (MS-34CT - internal reference CT) of the corresponding well site MS-34 target and internal reference was calculated, and the methylation rate was calculated as 1 / (2^dCT) × 100%. The inventors set up three technical replicates for each enzyme digestion period, calculated the mean measured methylation rate, and compared it with the expected methylation rate. The detection results and calculated methylation rate results for the 100%, 50%, and 0% methylation rate reference samples are shown in Tables 26-28, respectively. The average methylation rate distribution is as follows: Figure 10-12 .

[0185] Table 26 Detection Results of 100% Methylated Reference Standard

[0186]

[0187]

[0188] Table 27 Detection Results of 50% Methylated Reference Standard

[0189]

[0190]

[0191] Table 28 Detection Results of 0% Methylated Reference Material

[0192]

[0193] Test results showed that the consistency between the measured methylation rate and the expected methylation rate was ideal when the enzyme digestion time was 30-120 minutes; therefore, this time range was considered the optimal enzyme digestion time range. Enzyme digestion times shorter than 30 minutes did not allow for complete digestion, leading to an increase in background methylation rate and consequently affecting the specificity of the detection. When the enzyme digestion time exceeded 120 minutes, over-digestion (non-specific digestion) occurred, resulting in a lower measured methylation rate than expected, a weakened signal from the positive reference sample, and a decrease in detection sensitivity.

[0194] Example 6: Accuracy Study of Self-Prepared One-Step MSRE-qPCR Amplification System

[0195] In previous example studies, it has been confirmed that all three self-prepared reaction buffers meet the requirements for constructing a one-step MSRE-qPCR amplification system. To compare the accuracy of the three self-prepared reaction buffers in detecting methylation levels using the one-step MSRE-qPCR amplification system, the inventors prepared different one-step MSRE-qPCR amplification systems using these three self-prepared reaction buffers. These systems were then used to detect standards with methylation rates of 100%, 75%, 50%, 25%, 12.5%, 6.25%, and 0%, respectively. ACTB was used as an internal reference gene, and the measured methylation rate was obtained by calculating the MS-34dCT value. Finally, the measured methylation rate was divided by the theoretical methylation rate to calculate the cleavage efficiency of the unmethylated template. The reaction system with a cleavage efficiency close to 100% was considered the optimal one-step MSRE-qPCR amplification system. The specific implementation scheme is as follows:

[0196] 1. Test Sample Preparation

[0197] Mix MS-34 positive standards according to Table 29 Methylated HCT116g DNA (Takara, 3522) and leukocyte DNA with a known MS-34 methylation rate of 0% were used to prepare sufficient amounts of standards with gradient methylation rates of 100%, 75%, 50%, 25%, 12.5%, 6.25% and 0%.

[0198] Table 29 Dilution table of graded methylation standards

[0199]

[0200] 2. Preparation of the reaction system

[0201] Prepare the one-step MSRE-qPCR amplification system in the ice box according to Table 30. Place the reaction system on the heating module of the qPCR instrument, confirm the tube caps are sealed again, and then close the sample chamber door. Set the fluorescence channel selected for each detection tube according to Table 31, set the qPCR amplification program according to Table 32, and then start the detection.

[0202] Table 30 One-step MSRE-qPCR amplification system

[0203]

[0204]

[0205] Table 31 Fluorescence Channel Settings

[0206] Gene signal channel MS-34 FAM

[0207] Table 32 Reaction conditions for one-step MSRE-qPCR detection

[0208]

[0209] 3. After the program finishes running, export the detection data.

[0210] The measured methylation rate was obtained by calculating the dCT value (MS-34 CT value minus the internal control CT value) of the MS-34 target and internal control at the corresponding well positions. The calculation formula is: Measured methylation rate = 1 / (2^dCT) × 100%. During the experiment, the inventors set up three technical replicates for each methylation rate gradient standard. Based on the technical replicate experimental data, the mean measured methylation rate was calculated. Furthermore, the cleavage efficiency was obtained by dividing the mean measured methylation rate by the expected methylation rate. The measured methylation rate and its corresponding cleavage efficiency are detailed in Table 33. The data shows that the cleavage efficiency of the three self-prepared reaction buffers for each methylation rate gradient standard is close to 100%, indicating that all three self-prepared reaction buffers are suitable for one-step qPCR reaction systems.

[0211] Table 33 Detection results and enzyme digestion efficiency

[0212]

[0213]

[0214]

Claims

1. A nucleic acid detection kit, comprising a modification-sensitive restriction endonuclease and deoxyribonucleoside triphosphate. (dNTP), uracil-DNA glycosylase (UDG), primers that specifically recognize and / or bind to the target sequence, and / or probes that specifically recognize and / or bind to the target sequence.

2. The methylation detection kit according to claim 1, wherein the dNTP includes dUTP, dTTP, dATP, dCTP and / or dGTP.

3. The methylation detection kit according to claim 2, wherein the ratio of dUTP to dTTP in the dNTP is 1:10 to 10:1, preferably 1:

1.

4. The methylation detection kit according to claim 3, wherein the ratio is a molar mass ratio or a mass ratio.

5. The methylation detection kit according to any one of claims 1-4, wherein the modification-sensitive restriction endonuclease is a methylation-sensitive restriction endonuclease (MSRE).

6. The methylation detection kit according to claim 5, wherein the methylation-sensitive restriction endonuclease is selected from one or more of the following group: BstUI, SmaI, HinP1I, AciI, HpaI, HhaI, HpaII, ApaI, AvaI, and HaeII.

7. The methylation detection kit according to claim 5, wherein the methylation-sensitive restriction endonuclease is selected from one or more of the following: AciI, HpaII, HhaI, and HaeII.

8. The methylation detection kit according to any one of claims 1-7, wherein the concentration of the methylation-sensitive restriction endonuclease is 0.01-0.4 units / μL.

9. The methylation detection kit according to any one of claims 1-8, further comprising a nucleic acid polymerase and Mg... 2+ and / or buffer solution.

10. The methylation detection kit according to claim 9, wherein the buffer is a PCR amplification buffer.

11. The methylation detection kit according to claim 9, wherein the buffer is a buffer suitable for the DNA polymerase to exert its activity.

12. The methylation detection kit according to claim 9, wherein the Mg 2+ The concentration is 1–10 mM.

13. The methylation detection kit according to claim 9, wherein the pH of the buffer solution is from 7.0 to 9.

0.

14. The methylation detection kit according to any one of claims 1-13, further comprising primers that specifically recognize and / or bind to an internal reference gene and / or probes that specifically recognize and / or bind to an internal reference gene.

15. A method for detecting methylation, comprising: (a) Mixing a nucleic acid sample with the substance in the methylation detection kit according to any one of claims 1-13 to obtain a mixture; (b) The mixture is kept under conditions that allow the modified-sensitive restriction endonuclease to exert its digestive activity for 25-120 min to obtain the digestion product; (c) Amplification; (d) Obtaining test data and data analysis.

16. The methylation detection method according to claim 15, wherein the conditions under which the modification-sensitive restriction endonuclease exerts its enzymatic activity include a temperature of 37°C.

17. The methylation detection method according to claim 15 or 16, wherein the amplification comprises heating the mixture to above 85°C and holding for at least 5 minutes.

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