WO2020019700A1 - Detection reagent, kit and method for dna methylation - Google Patents

Abstract

A detection reagent, kit and method for DNA methylation, especially a detection reagent, kit and method for DNA methylation without CpG sites at flanking sequences. The reagent, the kit and the method employ a Taqman probe binding to a minor groove binder (MGB) and can determine the methylation of a CpG Island (CGI), and can determine isolated CpG sites in a CpG open sea and the like in a genosome without CpG at two flanking sequences. The reagent, the kit and the method are specific and sensitive, simple to operate, and do not require a control reaction or a fully-methylated standard DNA substance as a reference, thereby overcoming defects resulting from the control reaction and the fully-methylated standard DNA substance. In addition, the reagent, the kit and the method have higher repeatability and accuracy than the existing MethyLight technology.

Description

DNA methylation detection reagent, kit and method Technical field

The invention belongs to the technical field of analysis methods, and relates to a DNA methylation detection reagent, a kit and a method, in particular to a DNA methylation detection reagent, a kit and a method for a flanking sequence without a CpG site.

Background technique

Cytosine-5 DNA methylation (m5C) present in cancer tissues is considered to be an apparent DNA modification with potential clinical value [1]. In vertebrates, m5C occurs mainly in CpG dinucleotides. It has been confirmed in a variety of tumors that abnormal methylation of the CpG island (CGI) of the oncogene promoter leads to transcription inactivation [2]. However, CGI in the promoter represents only a small part of methylation, and CpG, which is mainly located in the genome, represents the most conserved DNA methylation target in eukaryotes, but the methylated region in this region The function is unclear. Recent studies have revealed the synergistic effect of methylation of non-promoter regions (such as genomic and UTR) on gene expression, and genomic methylation may be a potential therapeutic target in cancer [3]. In addition, genomic methylation may be a potential mechanism for RNA alternative splicing regulation [4], and can limit transcription initiation, thereby preventing abnormal transcription [5]. In previous studies, we found that differential methylation positions (DMPs) in patients with relapsed and non-relapsed colorectal cancer by EPIC methylation chips are rare in CGIs and promoters, but in open genes and genes. A lot in the body. We also found a set of hypermethylated CpG sites in the genome that are associated with gene overexpression.

Because of the great value of analyzing methylation in the genome, many methods and techniques for detecting methylation have been developed. Methylation-specific PCR (MSP) is an endpoint analysis technique [6,7]; after that, the second generation of PCR-based technology-MethyLight (quantitative MSP) was used for quantitative detection [8] -10]. MethyLight technology based on the AluC4 control reaction has been widely used to detect CGI methylation in tissue samples [11,12]. However, all these existing DNA methylation detection techniques require that the site to be tested is located in a region with clustered CpG dinucleotides, such as CpG islands, to allow the primers and probes designed to cover enough CpG sites It is not this site that is detected, but the state of co-methylation of all CpG sites covered by primers and probes. However, the prior art has the following problems:

(1) It is impossible to accurately detect the heteromethylation present in the genome. If adjacent CG sites do not exhibit co-methylation, the primers and probes required for qMSP technology cannot be successfully designed in this region.

(2) It is not possible to detect methylation levels outside the CpG island. The region outside the CpG island in the genome includes regions such as open sea and CpG shore, which are mainly located in the gene body and intergenic regions. There are fewer CG sites in this region, and there are few adjacent CG sites. Design primers and probes required for qMSP technology.

(3) The absolute quantification of the methylation level depends on the methylation standard. If you want to obtain the absolute value of the methylation level at a certain site, you must set the 100% methylation standard at the same time, and calculate it by the ratio of the sample and the standard. Percentage of methylation (PM). If the methylation standard production batches are different or there are unavoidable quality defects, such as the CG site is not 100% methylated, the spontaneous deamination of m5C is converted to C, and the DNA is degraded, the absolute value of methylation level will be quantified Serious errors.

The current methylation detection is for the detection of methylation sites on CpG islands. Such sites can only be detected unless sequencing (bisulfite pyrosequencing) is used, but it is not used in large sample cohort verification. Cost-effectiveness.

However, neither MSP nor MethyLight can analyze the isolated CpG sites flanking the CpG sites, which are mainly distributed in open regions of the gene.

Summary of the Invention

The purpose of the present invention is to provide a methylation detection reagent, a kit and a detection method.

Another object of the present invention is to provide a DNA methylation detection reagent, a kit and a method for any CpG site on the entire gene sequence.

Another object of the present invention is to provide a DNA methylation detection reagent, a kit and a method for a flanking sequence without a CpG site.

Another object of the present invention is to provide a DNA methylation detection reagent, a kit and a method that do not require a methylation standard.

Another object of the present invention is to provide a DNA methylation detection reagent, a kit and a method with high accuracy, cheapness, convenience, and speed.

The above object of the present invention is achieved by the following technical means:

In one aspect, the present invention provides a method for detecting DNA methylation, the method comprising the following steps:

a) converting unmethylated cytosine bases to uracil while leaving methylated cytosine bases unchanged;

b) using at least one pair of primers and at least one pair of oligonucleotide probes covering the CpG site to be tested to amplify the DNA sample transformed in step (1); wherein the oligonucleotide probe binds to a small groove Binder (minor groove binder, MGB);

c) Analyze the amplified product, and analyze the methylation status of the DNA to be tested from the presence of the amplified product.

The method detects that the CpG site is a CpG site of a heteromethylated region, or an isolated CpG site flanked by CpG, or a CpG site of a co-methylated region, or a non-co-methylated region CpG site. As a preferred embodiment, the CpG site detected by the method of the present invention is an isolated CpG site flanked by CpG.

Alternatively, the CpG site is a CpG site outside the CpG island, or a CpG site within the CpG island.

Alternatively, the CpG site is located in a genome, an intergenic region or a promoter.

As a preferred embodiment, the CpG site is located in the CpG open sea, CpG shore, and CpG shelf of the genome or intergenic region.

Various existing methods for detecting DNA methylation, such as using MethyLight (methylation-specific quantitative PCR) to check methylation of CpG islands (CpG islands, CGI), are mainly based on CpG islands in the promoter region Co-methylation is common in adjacent CpG sites. The traditional PCR-based methylation detection technology is based on the assumption that all CG sites in the CpG island are methylated simultaneously or not at the same time, which is called a co-methylated region. The designed primers and probes cover multiple CG sites in this area, and the ratio of simultaneous methylation of these CG sites is detected, not a high-resolution detection technique for a single CG site.

Although genome-wide methylation sequencing technology revealed that co-methylation of CpG islands is widespread, CGI co-methylation in the promoter represents only a small part of methylation, and there are still many non-co-methylation The region of methylation, in which some adjacent CG sites are methylated, and some are unmethylated, are called hybrid methylated regions. Heteromethylated regions mainly exist in CpG open sea, shore, shelf, and some CpG islands. In addition, there are many isolated CpG sites that are not located in the CGI, but are located in the flanking sequences of the genome and intergenic regions without CpG. Unfortunately, existing PCR-based technologies cannot detect such hybrid methylated CpG sites or isolated CpG sites without CpG flanking sequences.

The isolated CpG sites without CpG flanks are generally CpG sites located outside the CpG island, such as CpG opensea, CpG shore, and CpG shelf at the genome or intergenic region.

Wherein in step a), all unmethylated cytosine bases of the DNA sample are converted into uracil, while the methylated cytosine bases remain unchanged, and the unmethylated Cytosine bases are converted to uracil. The conversion agent is not particularly limited, and all the agents reported in the prior art that can achieve the conversion of cytosine to uracil may be, such as hydrazine, bisulfite, and bisulfite (such as sodium metabisulfite, Potassium bisulfite, cesium bisulfite, ammonium bisulfite, etc.). As an exemplary embodiment, the converting agent is selected from bisulfite.

Methylation occurs when a methyl group is added to cytosine. After treatment with a conversion agent such as bisulfite or bisulfite or hydrazine, unmethylated cytosine will become uracil because When performing amplification, uracil is similar to thymine and will be recognized as thymine. In the amplified sequence, cytosine that has not been methylated becomes thymine (C becomes T), and methylated cells Pyrimidine (C) does not change. Therefore, after transforming DNA samples with a transforming agent, the methylated CpG remains CpG (CG), while the unmethylated CpG is deaminated to TpG (TG) after conversion, and then a CG / TG-specific probe is used Binding CpG site.

Wherein, in step b), the length of the primer amplification is 50-200 bp.

Design primers with a length of 50-200bp, not only in high-quality DNA extracted from in vitro cultured cells or fresh frozen tissue, but also fragmented DNA extracted from formalin-fixed paraffin-embedded tissue (FFPE) In both cases, the real-time quantitative PCR can be effectively implemented, so that the methylation level of the sample can be sensitively detected. The length of the amplification is too long, not only the amplification efficiency of the PCR itself is rapidly reduced, but also the efficiency of the 5 'end of the probe to be cleaved by the DNA polymerase during the PCR process is reduced, which affects the accuracy of the detection; The primers of amplicons have high requirements on quality control standards such as DNA integrity. In common clinical samples such as FFPE, DNA is often highly fragmented.

As a preferred embodiment, the primer should avoid the CG site: this technique is particularly suitable for CG sites in the genome and intergenic regions, open seas and other regions, there are fewer adjacent CG sites, and the probe covers the CG site to be tested Primers should avoid other CG sites to avoid the effect of hybrid methylation on the detection results.

Wherein, in step b), one of the pair of oligonucleotide probes covering the CpG site to be tested specifically binds the CG sequence and the other specifically binds the TG sequence, that is, the pair of probes. Among them, one for binding methylated CpG sites and one for binding unmethylated CpG sites.

As a preferred embodiment, in the present invention, the pair of oligonucleotide probes covering the CpG site to be detected is a pair of Taqman probes. The 5 'end of each Taqman probe is connected to a fluorophore, and the 3' end is connected to a quencher and MGB group. And the fluorophores attached to the 5 'ends of the two Taqman probes have different emission light wavelengths. When the CpG site is completely matched, the probe structure is destroyed, and the fluorescence is detected. More specifically, one of the probes is a TG-Taqman-MGB probe directed to an unmethylated CpG site, and the other is a CG-Taqman-MGB probe directed to a methylated CpG site.

As a preferred embodiment, the fluorescent group is not particularly limited, and may be selected from the fluorescent groups for probes in the prior art. For example, for each probe, it may be selected from, for example, FAM, VIC, ROX, TAMRA, One of SYTO9, JOE / TET / HEX, Texas Red, NED / BODIPY / TMR-X, etc., but it is necessary to maintain a pair of probes, each of which has a different fluorophore with a different emission The wavelength of light so that they can be distinguished during fluorescence detection. It should be noted that FAM and SYTO9 have the same emission light wavelength, VIC and JOE / TET / HEX) have the same emission light wavelength, ROX and Texas Red have the same emission light wavelength, TAMRA and NED / BODIPY / TMR-X With the same emitted light wavelength, the selection of the above-mentioned fluorophores with the same emitted light wavelength should be avoided. Amplification is shown by the increase and decrease of fluorescence, and the increase and decrease of fluorescence is also directly used for analysis, and the methylation status of the DNA under study can be inferred from the fluorescence signal.

As a preferred embodiment, the quencher is not particularly limited, and the quencher of the fluorescent group in the prior art may be used, such as one of NFQ, BHQ1, and BHQ2.

In the Taqman probe used in the present invention, the MGB group connected at the 3 'end can increase the annealing temperature of the probe.

The main application of MGB is genotyping of DNA polymorphisms. In the present invention, MGB is introduced into the probe in the methylation detection, and the characteristics of the single base mismatch close to MGB, which makes the annealing temperature up to 10 ° C or more, such as 17 ° C, can be fully utilized in a simple way. The methylated and non-methylated CG sites of the location are designed in the region of the probe near the MGB, that is, the detection of non-CpG island methylation sites in the prior art has been overcome for many years.

The present invention finds that if the MGB is not connected and the probe annealing temperature reaches 70 ° C, the probe must be very long, especially in non-CpG island areas, where the CG content is low and may grow to 40bp. In such a long probe, the only CG / TG mismatch difference has almost no effect on the annealing temperature, so CG / TG cannot be identified.

As a preferred embodiment, the number of probe bases is reduced as much as possible: the probe annealing temperature should be 10 degrees higher than the primer annealing temperature to ensure that the binding of the probe to the DNA template occurs before the primer annealing extension. The shorter the probe, the more specific the probe is to identify methylated and unmethylated CG sites. The length of the probe should be controlled between 10 and 20 bp. As a more preferred embodiment, the length of the probe should be controlled between 12 and 18 bp.

As a preferred embodiment, the CpG site to be detected should be located as far as possible in the region 3 'to the middle of the probe. As a more preferred embodiment, the CpG site is located in a third region near the 3 ′ end of the probe.

Compared with the perfect pairing, the single base mismatch in the 3 ′ half region (MGB region) of the probe has an annealing temperature difference of up to 17 ° C, while the single base mismatch in the 5 ′ end region can only produce 2 ～ 10 ° C annealing temperature difference. Therefore, the probe at the 3 'end of the CG site to be detected has higher specificity.

As a preferred embodiment, the 5 'end of the probe should be close to the 3' end of the forward primer, but should not overlap with the forward primer, and can be spaced more than 1 base from the 3 'end of the forward primer. The probe may overlap the reverse primer.

Since the exonuclease activity of the DNA polymerase is maximal in the initial stage of DNA synthesis chain extension, the 5 'end of the probe should be as close as possible to the forward primer, which can ensure that the fluorescent group coupled to the 5' end of the probe can extend to the maximum It is cleaved by DNA polymerase and releases fluorescence to ensure the accuracy of detection. In addition, when the polymerizability and exo-activity of the DNA polymerase are maximized, the probe bound to the template strand can be efficiently cleaved to avoid premature termination of DNA extension and inaccurate detection.

The invention ingeniously designs primers and probes, and adopts MGB-binding probes. After genomic DNA is treated with a transforming agent, unmethylated cytosine is converted to uracil (UpG), and methylated cytosine residues Unaffected (mCpG), which results in methylation-dependent sequence differences in genomic DNA. During the PCR amplification process, fluorescently labeled TaqMan MGB probes with different emission wavelengths were used to distinguish single-base differences from transformant treatment. In the same reaction system, the same pair of primers are used to amplify methylated and unmethylated alleles. Sequence discrimination only occurs during the process of hybridization of fluorescent probes. This distinction is based on the complete sequence pairing and error The matching annealing temperature difference (Figure 1).

The present invention uses a fluorescently labeled MGB probe that emits light wavelengths to specifically bind methylated allele sequences, and uses another fluorescently labeled MGB probe that emits light wavelengths to specifically bind unmethylated alleles sequence. The single base mismatch in the 3 'half region (MGB region) of the probe has an annealing temperature difference as high as 17 ° C compared to the perfect pairing. This allows us to design probes to cover only a single CpG dinucleotide, which can measure the methylation level of isolated CpG.

In addition to detecting methylation of a single isolated CpG site, the method of the present invention can simultaneously detect methylation of multiple isolated CpG sites. At this time, for each CpG site, a pair of primers and a pair of oligonucleotide probes covering the CpG site to be tested need to be designed. In addition, the primer amplification regions of each CpG site to be tested do not overlap to avoid competition in the multiplex PCR process, and the fluorophore attached to the 5 'end of each probe has a different emission light wavelength. In addition, the compatibility of multiple qMSP primers and probe combinations needs to be analyzed: After the primer probe design is completed, the secondary structure that may exist between all primer probes in the multiple qMSP system should be theoretically excluded first.

(http://www.cstl.nist.gov/strbase/AutoDimerHomepage/AutoDimerProgramHomepage.htm), determine primer primer combinations for multiple qMSPs.

In step c), the methylation state in the DNA sample to be tested is analyzed by the methylation percentage parameter PM. In the prior art, the calculation method of the methylation percentage parameter PM is: (methylated fluorescence value / internal reference fluorescence value) _{test sample} / (methylated fluorescence value / internal reference fluorescence value) _{fully methylated standard}

In the present invention, the methylation percentage parameter PM is:

Methylation / (methylation + unmethylation) × 100%;

Specifically:

Methylated fluorescence value / (methylated fluorescence value + unmethylated fluorescence value) × 100%;

More specifically:

100 / (1 + 1/2 ^-ΔCT ), ^ΔCT = CT _{methylated fluorescence–} CT _{unmethylated fluorescence}

It can be seen from the calculation method that compared with the calculation method used in the traditional technology, the present invention more directly expresses the methylation ratio of the sample to be measured, and does not need to use an internal reference reaction and a fully methylated standard, which is more convenient. At the same time, errors caused by the use of internal reference reactions and fully methylated standards are also avoided.

In another aspect, the present invention also provides a reagent for detecting DNA methylation, the reagent contains at least one pair of oligonucleotide probes, the probes cover a CpG site, and the probes It is a probe connected to MGB.

The reagents of the invention do not contain fully methylated standards.

Among them, one pair of oligonucleotide probes specifically binds the CG sequence and the other specifically binds the TG sequence.

As a preferred embodiment, a pair of oligonucleotide probes in the reagent is a pair of Taqman probes, and a 5 'end of each Taqman probe is connected to a fluorophore, and a 3' end is connected to a quencher and an MGB group. And the fluorophores connected at the 5 'ends of the two Taqman probes have different emission wavelengths.

Further, the fluorophore may be selected from any two of FAM (or SYTO9), VIC (or JOE / TET / HEX), ROX (or Texas Red), TAMRA (or NED / BODIPY / TMR-X) Species.

Further, the quencher is selected from one of NFQ, BHQ1, BHQ2, and the like.

As a preferred embodiment, the length of the probe is between 10 and 20 bp; more preferably, the length of the probe is between 12 and 18 bp.

As a preferred embodiment, the CpG site to be tested is located in a region near the 3 'end of the probe; preferably, the CpG site is located in a third region near the 3' end of the probe.

As a preferred embodiment, the reagent further includes at least a pair of primers; the pair of primers corresponds to a pair of probes.

A pair of primers includes a forward primer and a reverse primer, and a pair of probes includes a CG-binding probe and a TG-binding probe. The pair of probes must be located in the amplicon region of the pair of primers. Consider keeping the probe as close as possible to the forward primer, but not overlapping it. This technology can be applied to multiple PCR systems, using two or more pairs of primers and two or more pairs of probes corresponding to them respectively, to simultaneously detect two or more test sites in a reaction system. Methylation level.

As a preferred embodiment, the length of the primer amplification is 50-200 bp.

As a preferred embodiment, the 5 'end of the probe is close to the 3' end of the forward primer.

Optionally, the reagent further contains a transforming agent, and the transforming agent is selected from one or more of hydrazine, bisulfite, and bisulfite; preferably, the transforming agent is selected Since bisulfite.

Optionally, the reagent further contains one or more of a DNA polymerase, dNTPs, Mg ²⁺ ions, and a buffer solution; preferably, the reagent contains a DNA polymerase, dNTPs, Mg ²⁺ ions And buffer.

In the reagent of the present invention, when there are multiple pairs of primers and multiple corresponding probes, the amplification regions of each pair of primers do not overlap, and between the primers and the primers, between the probes and the probes, and between the probes and the Primers should be compatible with each other without secondary structures such as dimers and hairpin-like structures to ensure the efficiency and specificity of multiplex PCR amplification.

In another aspect, the present invention also provides a kit for detecting DNA methylation, the kit containing the above-mentioned reagent for detecting DNA methylation.

As an optional embodiment, the kit of the present invention includes one or more containers divided into receiving reagents therein. For example, a first container containing a probe that specifically binds to a CpG site; a second container containing an amplification primer; a third container containing a conversion agent that sensitively converts unmethylated cytosine, and the like.

In another aspect, the invention also provides a system for detecting DNA methylation, the system comprising:

a) DNA methylation detection building block; and,

b) Output component.

The DNA methylation detecting member contains the above-mentioned reagent or kit.

The output component is used for outputting methylation ratio or methylation percentage parameter of methylation, PM.

In another aspect, the present invention also provides applications of the above-mentioned DNA methylation detection method, reagent, kit, and system in disease diagnosis.

The disease is a methylation-related disease.

Alternatively, the diseases include but are not limited to: tumors; CNS dysfunction and injury; brain injury; mental disorders; dementia; cardiovascular diseases; gastrointestinal diseases; respiratory diseases; inflammation, infection, immunity; skin, muscle, Connective tissue or bone disease; endocrine and metabolic diseases; headache or sexual dysfunction, etc. It has been reported in the prior art that the occurrence of DNA methylation may be related to various diseases [18-36].

The tumor includes, but is not limited to, colorectal cancer, breast cancer, lung cancer, prostate cancer, liver cancer, gastric cancer, esophageal cancer, pancreatic cancer, nasopharyngeal cancer, thyroid cancer, kidney cancer, bladder cancer, cervical cancer, ovarian cancer, Nervous system tumors, lymphomas, and leukemias.

Promoter methylation of tumor-related genes is an important indicator of cancer and can therefore be used in many applications, including cancer diagnosis and early detection, estimation of cancer development risk, cancer prognosis, follow-up examination after treatment, and anti-cancer Estimate of treatment response. Recently, active attempts have been made to examine the methylation of promoters of tumor-related genes in blood, sputum, saliva, stool or urine, and the results have been used in the diagnosis and treatment of various cancers.

Of course, the method, reagent and kit for detecting DNA methylation of the present invention can also be used for non-disease diagnostic applications, such as, but not limited to, cell line authentication and organization using species methylation characteristics as markers Source identification, prenatal diagnosis, microbial identification, germline identification of new species, etc. The application of methylation as a marker has been reported in the prior art [37-46].

On the other hand, the present invention also provides a method for diagnosing a disease. The method is to detect the DNA methylation status of a sample to be tested by using the methods, reagents, kits, and systems described above, and determine whether the DNA methylation status is determined based on the DNA methylation status. Sickness, risk, probability of illness, course of illness, type of illness, etc.

In another aspect, the present invention also provides a disease diagnosis system, which includes the above-mentioned system for detecting DNA methylation.

As a preferred embodiment, the disease diagnosis system further includes a result judgment component.

The result judging component is used to compare the methylation results of the sample to be tested with the normal sample, so as to analyze whether the disease, the risk, the probability of the disease, the course of the disease, the type of the disease, etc. One or more. Further, the result judgment component compares the methylation status of the test sample and the normal sample, and outputs whether or not the disease is present, the risk of the disease, and the probability of the disease according to the difference or difference between the test sample and the normal sample. The course of the disease, the type of disease, etc.

The beneficial effects of the present invention:

1. Can detect the methylation level of a single CpG:

The present invention proposes a novel QASM (quantitative analysis of single-CpG methylation) technology for the first time, which uses a probe combined with a minor groove binder (MGB) and uses a real-time quantitative PCR The accurate quantification of the methylation level of a single CpG solves the problem that traditional qMSP cannot quantify the methylation of CG sites outside the CpG island.

2. Better repeatability and accuracy:

First, the detection design of this technology is simpler and does not require an independent input reference response, so the inevitable deviations and errors from the input reference response will not accumulate and will not affect the final result. As shown in Figure 2E, the standard deviation of the two independent measurements is significantly smaller than the standard deviation using the Alu-C4 response as the input reference.

Secondly, because the PM of this technology is determined by the ratio of methylation / (methylation + unmethylation), it is not necessary to calculate fully methylated DNA as a reference. If the reference DNA is not fully methylated, the PM determined by the ratio of the test sample to the reference DNA is obviously incorrect, which also explains why the results of this technique and pyrosequencing are almost identical, while traditional MethyLight The results of phosphate sequencing are linearly related, but there is a certain bias in PM.

Since the present invention is a high-resolution detection technique for a single CG site, which is much higher than the low resolution of 3 to 10 CG sites in the conventional technique, the present invention can effectively detect the presence of heterogeneous methyl groups in the methylation group. The methylation level of the CG site in the glycosylated region and the isolated CpG site flanked by CpG.

Finally, the single CpG resolution of this technique makes the results unaffected by the methylation variation of CpGs in the flanking sequences. Therefore, the PM measured by this technique is almost the same as the methylation percentage determined by pyrosequencing.

3. Cheaper, more convenient and faster:

It is worth noting that by comparing with bisulfite pyrosequencing, which is widely regarded as the most reliable methylation determination method, the accuracy of the present invention is consistent with that of pyrosequencing, and very accurate methylation can be achieved.化 Quantitative. This high-resolution methylation information was previously only available using bisulfite genome sequencing or bisulfite pyrosequencing, but this is true for large samples of DMPs in formalin-fixed paraffin-embedded tissue. For queue verification, there is no cost-effective advantage or feasibility. However, the method of the present invention is cheaper, more convenient, and faster than pyrosequencing when its accuracy is consistent with that of pyrosequencing. Pyrosequencing is the gold standard technique for detecting DNA methylation levels and can also be used to detect methylation levels of isolated CG sites targeted by this technology. However, pyrosequencing requires a special pyrosequencer, which is difficult to obtain (generally not available in laboratories), expensive sequencing (about 300 / sample), and long sequencing cycle (about 1 month); this technique requires only one routine The real-time PCR instrument is cheap, and the data can be obtained immediately.

4. Wide range of application, methylation detection can be achieved for both CGI and non-CGI single CpG sites:

The traditional MethyLight technology is based on the assumption that all CpGs of CGI are methylated or unmethylated at the same time, and the percentage of co-methylation of all CpG sites covered by the primer probe is determined. However, if the CpGs covered by the primers and probes in the test site and flanking sequences are not co-methylated, the original technology will not be able to accurately check the methylation status of the test zone. Because this technique shows a high degree of sensitivity, specificity and accuracy for a single CpG measurement. The method of the present invention is used to determine the methylation level of a single CpG dinucleotide, but it is not limited to open seas with flanking sequences lacking CpG. The method of the invention can more accurately detect the methylation level of the CGI test area.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the detection principle of the present invention.

2 is the specificity, sensitivity, repeatability and quantitative accuracy of the present invention;

A. Use this technology to detect the methylation levels of FAT3, FHIT and KIAA1026 in fully methylated and unmethylated DNA;

B. After using DNA demethylation reagent 5-aza-2'-deoxycytidine (5-aza) to treat HCT116 cells; the methylation levels of FAT3, FHIT and KIAA1026 detected by this technology were reduced as expected;

C. Repeatability of this technique on tumor heterogeneous samples;

C1. Repeatability of FAT3 on tumor heterogeneous samples;

C2. Repeatability of FHIT on tumor heterogeneous samples;

C3. Repeatability of KIAA1026 on tumor heterogeneous samples;

D. Test range and quantitative accuracy of this technology by standard curve;

E. Compare repeatability with techniques using Alu-C4 reaction correction.

Figure 3 is a quantitative comparison of methylation quantification accuracy of FAT3, FHIT and KIAA1026 with bisulfite pyrosequencing;

A. Assess the quantitative accuracy of methylation at the FAT3 site for comparison with bisulfite pyrosequencing;

B. Assess the quantitative accuracy of methylation at the FHIT site for comparison with bisulfite pyrosequencing;

C. Evaluate the quantification accuracy of methylation at KIAA 1026 for comparison with bisulfite pyrosequencing.

Figure 4 shows the results of screening EPIC microarrays in 45 tumor samples using this technique.

FIG. 5 compares the accuracy of the PM calculation method (A) of the present invention with the traditional PM calculation method (B).

detailed description

The technical solutions of the present invention are further described below through specific examples. The specific examples do not represent a limitation on the protection scope of the present invention. Some non-essential modifications and adjustments made by others according to the concept of the present invention still belong to the protection scope of the present invention.

In the present invention, "CpG site of a heteromethylated region" and "CpG site of a non-co-methylated region" mean one. For example, there are 10 CpGs in a 100bp DNA region. The epigenetics community usually assumes that these 10 CpGs are methylated or unmethylated at the same time, which is called a "co-methylated region". The genome Many regions are such "co-methylated regions". The traditional technology is based on such a high probability "co-methylated" phenomenon. Primers and probes are designed to cover multiple CpG sites in a 100 bp region. What is actually detected is The proportion of cells with multiple CpG sites simultaneously methylated in this region. However, in recent years, high-throughput sequencing studies have found that there are many adjacent CpG sites in the genome that are not methylated or unmethylated at the same time. They are called "CpG sites in hybrid methylated regions" or "CpG sites in non-co-methylated regions" [47-48].

Genome: A gene is all the nucleotide sequences required to produce a polypeptide chain or functional RNA. A gene body is the main part of a gene. It usually refers to a gene that removes its promoter region (usually refers to the upstream and 2000bp region downstream).

Intergenic region: The intergenic region refers to the spacer sequence between genes and is a segment in the genome that does not have a genetic effect and does not belong to the genetic structure.

CpG islands: The distribution of CpG dinucleotides in the human genome is very uneven. In certain sections of the genome, CpG remains at or above the normal frequency. CpG islands are mainly located in the promoter and exon regions of the gene, and are some regions rich in CpG dinucleotides, with a length of 300-3000bp. It is usually defined as GC content exceeding 55%, and the ratio of actual to expected CpG dinucleotide numbers is greater than 65%. The expected CpG dinucleotide number calculation method is (C number * G number) / sequence length.

CpG shore: In the areas immediately adjacent to the CpG island, the length is about 100-3000 bp. The frequency of CpG dinucleotides does not meet the requirements of the CpG island definition, but it is higher than other regions of the genome. Compared to the CpG island, These immediate flanking regions are called "shore" CpG islands of CpG islands: In regions of the genome that are far away from CpG islands, the frequency of CpG dinucleotides is much lower than that of CpG islands. Compared to CpG islands, these genomes are the most extensive Area is called "open sea"

CpG shelf: In the areas on both sides of CpG shore, the frequency of CpG dinucleotides is lower than shore, but higher than open sea. Compared to CpG islands, "shores" and the broad "high seas", these are far from CpG islands and The area closer to the "shore" is called the CpG "shelf"

Flanking sequences do not have isolated CpG sites for CpG: In the CpG open sea and shore regions, CpG dinucleotides appear less frequently, and CG sites are often isolated, often lacking in 100-200bp PCR amplified sequences on their sides. Other CpG sites.

Tissue DNA methylation level has potential clinical application value. There are various methods for detecting DNA methylation, such as using MethyLight (methylation-specific quantitative PCR) to check the methylation of CpG islands (CGI). However, the CGI in the promoter represents only a small part of the methylation group, and it is now gradually found that CpG, which is mainly located in the genome, plays an important role in the occurrence and development of disease and abnormal intracellular molecular events. Unfortunately, existing PCR-based techniques cannot detect such isolated CpG sites without CpG on both sides of the sequence. Therefore, the present invention provides a new QASM analysis method, which uses a Taqman probe bound to a minor groove binder (MGB).

Specifically, after genomic DNA is treated with a transforming agent, unmethylated cytosine is converted to uracil (UpG), while methylated cytosine residues are not affected (mCpG), which results in the production of formazan in genomic DNA. Glycation-dependent sequence differences. We used two different fluorescently labeled MGB during PCR amplification

Probe to distinguish single base differences from bisulfite treatment. In the same reaction system, the same pair of primers are used to amplify methylated and unmethylated alleles. Sequence discrimination only occurs during the process of hybridization of fluorescent probes. This distinction is based on the complete sequence pairing and error The matching annealing temperature difference (Figure 1).

The present invention utilizes the 5 'exonuclease activity of a DNA polymerase to cleave a dual-labeled probe, and hybridizes with the CG / TG sequence of the DNA treated by the transforming agent. Cleavage of the 5 'exonuclease separates the 5'-fluorophore from the 3'-quenching agent, releasing the fluorophore and generating a detectable fluorescent signal. Minor groove binding (MGB) binding to 3'-quencher allows the use of shorter probes, and has high sensitivity and specificity for single base mismatches. Methylated CpGs remain CpG during the conversion agent treatment, while unmethylene CpG is deaminated to TpG after conversion by the conversion agent. Therefore, using a CG / TG specific probe carrying two different fluorophores can When performing a one-step methylation detection, the single-base mismatch in the 3 'half region (MGB region) of the probe has an annealing temperature difference of up to 17 ° C compared to the perfect pairing. This allows us to design probes to cover only a single CpG dinucleotide, which can measure the methylation level of isolated CpG. PM is determined by the CT threshold ratio of the two fluorescent probes in the amplification cycle. The ratio between the signals from the methylated probe and the unmethylated probe can achieve accurate quantification of the methylation level of the test site. And no need to amplify control genes, such as Alu-C4 and ACTB, to reflect and correct the amount and integrity of the starting DNA template; in addition, it is no longer necessary to use CpGenome fully methylated DNA samples as a reference for calculating each sample PM samples.

The QASM detection method of the present invention is different from the traditional MethyLight in that it has a high resolution for detecting the methylation status in the genome and can detect the methylation level of a single CpG. This detection based on the ratio of methylated probes to non-methylated probes can improve the sample normalization method in PCR-based detection technology, because it does not need to use a positive control to calculate PM (fully methylated DNA), The total amount of control-corrected DNA template (Alu-C4PCR reaction) needs to be entered. Therefore, it is not susceptible to cancer-related copy number variation. This advantage is even more pronounced in complex clinical tissues with large samples.

We designed and applied this QASM technique to detect the methylation levels of three isolated CpG sites located in the CpG open sea of the FAT3, FHIT and KIAA1026 genomes. We evaluated the results of this QASM technique in tumor DNA samples by comparison with "gold standard" bisulfite pyrosequencing. In addition, we use the methylation / unmethylation ratio to calculate PM, thereby avoiding the need to add another independent PCR reaction to correct the total amount of DNA template from different samples. We have also developed a calculation method that allows researchers to determine the percentage of methylated alleles in a PCR reaction without using a fully methylated DNA sample as a control. We also described the PCR conditions in this technique and how to design primers and paired probes to determine the methylation level of isolated CpG faster and more accurately.

This technology is not only very specific and sensitive, but also easy to operate. It does not require control reactions and fully methylated DNA standards as references, which can overcome their shortcomings. In addition, it has higher repeatability and accuracy than existing MethyLight technology. We used this technique to determine three isolated CpG loci located in CpG open sea in 45 colorectal cancer samples, and found that this method can be used to quantitatively detect methylation in clinically complex samples.

The QASM detection method is technically sufficient to reliably detect methylation levels in isolated CpG sites. If suitable primers and probes are designed, they can also be used to more accurately detect the methylation level of CGI.

material

Fresh frozen tumor tissue samples were obtained from 45 patients with primary colorectal adenocarcinoma. The patients included 32 men and 13 women. Of these patients, 17 were stage I tumors and 28 were stage II tumors; 18 patients had recurrence at follow-up and 27 patients had no recurrence at follow-up. See Table 1 for details.

Table 1 Tumor samples

Example 1 Method for quantifying DNA methylation at CpG sites

DNA isolation and bisulfite conversion

Using QIAamp DNA Mini Kit (Qiagen, 51306) and EZ DNA methylation kit (Zymo Research, D5002), the genomic DNA in the above samples was extracted and bisulfite modified according to the instructions [7,14].

QASM detection

After bisulfite conversion, genomic DNA was amplified by real-time quantitative PCR. In short, bisulfite-converted genomic DNA was amplified using primers and a pair of oligonucleotide probes covering the CpG site to be tested, with a fluorescent report attached to the 5 'end of each oligonucleotide probe The dye 6FAM or VIC (specifically binds the CG sequence and the TG sequence, respectively), is coupled to the quencher-MGB group (MGB-NFQ) at the 3 'end (Figure 1). Taq DNA polymerase will cleave the probe and release the reporter gene during 5 'to 3' exonuclease activity during DNA extension. Its fluorescence can be detected by the Applied Biosystems QuantStudio 7Flex real-time PCR system. The initial DNA template concentration can be obtained from the CT (cycle threshold) value of the fluorescent signal [15]. The fully methylated and completely unmethylated standard samples were mixed at a certain ratio and then tested by QASM to draw a standard curve. We use a 20uL reaction system, which includes 500nM primers, 150nM probes, 200nM each of dATP, dCTP, dGTP, and dTTP, 2.25mM MgCl ₂ , 0.75U HotStar Taq enzyme, 1X PCR buffer. The reaction conditions were: DNA after bisulfite conversion at 95 ° C for 15 minutes, then 50 cycles of 94 ° C for 30 seconds, 56-60 ° C for 1 minute, and 72 ° C for 1 minute.

Primer and probe sequences

Aiming at the three sites to be tested in the FAT3 (gene ID: 120114), FHIT (gene ID: 2272) and KIAA1026 (gene ID: 23254) genes, we designed three sets of primers and probes specifically used in the present invention ( Table 2). The 200bp flanking sequences of the three isolated CpG sites to be tested are shown in Table 3.

Table 2 Primer and probe sequences used in the present invention

Table 3. Flanking sequences of three isolated CpGs

Methylation percentage calculation

The QASM test data of the CpG site to be tested is expressed in PM, but the calculation method is different from the previously reported method [8]. In the method of the invention, the PM of each sample is equal to methylation / (methylation + non-methylation) × 100. For specific calculation, we use the following formula: PM = 100 / (1 + 1/2 ^{-ΔCT )} , ΔCT = CT _FAM -CT _VIC

Methylation test results

The following Tables 4 to 6 show the detection results of FAT3, FHIT, and KIAA1026 in 10 clinical colorectal cancer tissues, respectively.

Table 4 FAT3 methylation test results

Table 5 FHIT methylation test results

Table 6 Methylation test results of KIAA1026

QASM technical verification

The inventors screened differentially methylated promoters (Differentially Methylated Positions, DMPs) between relapsed and non-relapsed colorectal cancer samples by EPIC microarray. DMPs are few in CGIs and promoters, but many in open seas and genomes. However, traditional MethyLight analysis cannot detect CpG methylation levels in CpG open seas without flanking CpG clusters. We selected three isolated CpG loci located in the open sea of the FAT3, FHIT and KIAA1026 genomes, and tested each of the techniques in human colorectal cell lines HCT116 and CpGenome methylated and unmethylated DNA (Millipore S7821 & S7822). Item properties.

Primers were first tested for specificity with methylated and unmethylated probes (Table 2). The PM of methylated DNA standards is close to 100%, consistent with its fully methylated state. In contrast, the PM of the unmethylated DNA standard was close to zero (Figure 2A). In addition, after treating HCT116 cells with DNA demethylation reagent 5-aza-2'-deoxycytidine (5-aza), the levels of FAT3, FHIT, and KIAA1026 methylation detected by this technology decreased as expected ( P <0.05, Figure 2B). This indicates that the method of the present invention can specifically distinguish methylated and unmethylated gene loci.

The reproducibility of this technique to detect methylation levels in 10 colorectal cancer DNA samples (Table 1) was further analyzed. Repeatability was tested by performing two independent qPCR reactions for each site tested. As shown in Figure 2C, the results of the three test sites are the CT _FAM- CT _VIC values obtained in two measurements, the standard deviation of the two independent measurements is low, and the delta of the first and second measurements is The CT value was linearly correlated with R ² > 0.999. Taken together, these results indicate that the technique can produce reproducible results from multiple measurements in complex clinically heterogeneous samples.

Finally, we test the detection range and quantitative accuracy of this technique with a standard curve. CpGenome universal methylated DNA and universal non-methylated DNA are mixed in different ratios. Use this technology to track the changes in methylation levels of FAT3, FHIT and KIAA1026 at different mixed ratio standards. The results show that even after 10,000-fold dilution of unmethylated DNA, methylation levels at three sites can still be reliably detected. We calculated the PM from each dilution to determine the quantitative accuracy of the technique, and the results showed that the methylation quantification of the technique on four orders of magnitude was linear (Figure 2D). These results indicate that the technique has high sensitivity and quantitative accuracy.

Example 2 Bisulphite Pyrosequencing Comparison and Evaluation Quantitative Accuracy

Pyrosequencing was used as a reference to further verify the accuracy of the methylation quantification method of the present invention. In 10 colorectal cancer tissues (Table 1), we used pyrosequencing and this technique to detect the methylation levels of the three CpG sites. The primers for pyrosequencing are shown in Table 2. The PM measured by the present invention is linearly correlated with the methylation percentage obtained by pyrosequencing (Figure 3; FAT3 is 0.9690, FHIT is 0.9954, and KIAA1026 is 0.8755, all P <0.001). Not only that, the methylation percentages of PM and pyrosequencing are surprisingly consistent. This is more accurate than the traditional MethyLight previously reported [11,12]. Therefore, this technique has the same quantitative accuracy as the bisulfite pyrosequencing method, and is simpler, easier to obtain, and cheaper to operate.

Example 3 Comparison of repeatability with the technique using Alu-C4 reaction correction

In the past, all qPCR-based detection techniques required internal reference genes for quantification. Traditional MethyLight widely uses AluC4 as a control reaction to evaluate and correct the total amount of starting DNA template [8,11,12]. This high-copy control amplicon is less susceptible to changes in cancer-related genes than other single-copy control genes. Unfortunately, the effects of genome-wide copy number variations and mutations on AluC4 stability cannot be completely avoided. The advantage of the present invention is that without such an internal reference reaction, each independent sample can obtain an accurate PM independent of the total amount of the starting DNA template. In addition, we compared the repeatability of the two detection methods in 10 clinical tumor samples (the specific steps of traditional MethyLight refer to Eads CA, Danenberg KD, Kawakami K, et al. MethyLight: a high-throughput assay to DNA measure methylation .Nucleic Acids Res. 2000.28 (8): E32.). We found that in the traditional MethyLight using the Alu-C4 reaction as the input control, the PM standard deviation of two independent measurements is higher than the PM standard deviation of the methylated / unmethylated signal ratio used in this technique to correct the input difference (Figure 2E). The higher standard deviations observed in traditional MethyLight may come from random PCR at low template concentrations [16] and the accumulation of bias in two independent reactions. However, this technique is less likely to be affected by these factors because the signals from methylated and unmethylated probes are generated using the same primers in the same reaction system.

Example 4 Comparison of EPIC microarray to evaluate the reliability of quantification

This technique was used to verify the methylation status of DMPs screened in the Illumina MethylationEPIC (EPIC) BeadChip microarray. EPIC array is the second-generation product of Illumina HM450microarray. Its detection probe coverage in CpG open sea has increased significantly, which provides a valuable tool for screening biologically and clinically significant isolated CpGs [17] . Forty-five colorectal adenocarcinoma tissues were used for this experiment (Table 1). We use this technique to detect and compare the methylation percentages of cg00561674 (FAT3), cg05704547 (FHIT) and cg06887407 (KIAA1026) in the EPIC microarray to determine the reliability of the EPIC microarray. EPIC microarray (EPIC microarray technology refers to Pidsley, R, Zotenko, Peters, TJ, et al. Critical evaluation of the Illumina Methylation EPIC BeadChip microarray for whole-genome DNA methylation, profiling. Genome, Biol.208.17: 1 detection The relative methylation level (β value) has a good linear correlation with the PM obtained by this technology (cg21101720R = 0.9208; cg21101720R = 0.29790; cg14215472 0.80.8. All P <0.001) (see Figure 4).

Embodiment 5 Accuracy Comparison between the PM Calculation Method of the Present Invention and the Traditional PM Calculation Method

In 10 clinical tumor samples, we compared the PM value of the KIAA1026 locus obtained by this technology calculation system and the traditional technology calculation method, and compared it with the pyrosequencing results. The experimental and calculation methods of this technology group have been described above; in the traditional technology group, a fully methylated standard must be added as an external reference, and two independent qPCR reaction systems need to be set up for each sample to detect the methyl groups to be tested respectively. The total amount of reference genes in AluC4 and AluC4 was calculated as follows: (methylated fluorescence value / internal reference fluorescence value) _{test sample} / (methylated fluorescence value / internal reference fluorescence value) _{fully methylated Standard} .

Compared with the results of pyrosequencing, although the PM obtained by the computing system of this technology and traditional technology is well correlated with the methylation ratio obtained by pyrosequencing, the linear correlation of this technology is stronger than that of traditional technology (Figure 5A vs FIG. 5B, R = 0.8412VS.0.7698). In addition, in traditional MethyLight technology using AluC4 and fully methylated standards as internal and external references respectively, the calculated PM value is far less consistent with the methylation ratio obtained by pyrosequencing (Figure 5A) vs. Figure 5A).

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Claims (22)

1. A method for detecting DNA methylation, which comprises the following steps:

   a) converting unmethylated cytosine bases to uracil while leaving methylated cytosine bases unchanged;

   b) using at least one pair of primers and at least one pair of oligonucleotide probes covering the CpG site to be tested to amplify the DNA sample transformed in step (1); wherein the oligonucleotide probes bind to MGB;

   c) Analyze the amplified product, and analyze the methylation status of the DNA to be tested from the presence of the amplified product.
2. The method according to claim 1, wherein the CpG site is a CpG site of a heteromethylated region, or an isolated CpG site flanked by CpG, or a co-methylated region CpG sites; or CpG sites in non-co-methylated regions;

   Or preferably, the CpG site is a CpG site outside the CpG island, or a CpG site inside the CpG island;

   Or preferably, the CpG site is located in a gene body, an intergenic region or a promoter;

   More preferably, the CpG site is located in the CpG open sea, CpG shore, CpG shelf of the genome or intergenic region.
3. The method according to any one of claims 1-2, wherein in step a), a conversion agent is used for the conversion, and the conversion agent is selected from the group consisting of hydrazine, bisulfite and bisulfite. One or more; preferably, the converting agent is selected from bisulfite.
4. The method according to any one of claims 1-3, wherein in step b), the length of the primer amplification is 50-200 bp.
5. The method according to any one of claims 1-4, wherein in step b), the pair of oligonucleotide probes covering the CpG site to be detected, one of which specifically binds to a CG sequence, and the other One specifically binds the TG sequence.
6. The method according to any one of claims 1-5, wherein the pair of oligonucleotide probes covering the CpG site to be detected is a pair of Taqman probes, and 5 'of each Taqman probe A fluorophore is connected to the end, a quencher and an MGB group are connected to the 3 ′ end, and the fluorophores connected to the 5 ′ ends of the two Taqman probes have different emission wavelengths.
7. The method according to any one of claims 1 to 6, characterized in that, for each Taqman probe, the fluorescent group is selected from the group consisting of FAM, VIC, ROX, TAMRA, SYTO9, JOE / TET / HEX, Texas Red And NED / BODIPY / TMR-X;

   Preferably, the quencher is selected from one of NFQ, BHQ1 and BHQ2.
8. The method according to any one of claims 1 to 7, wherein the length of the probe is between 10 and 20 bp; preferably, the length of the probe is between 12 and 18 bp.
9. The method according to any one of claims 1 to 8, characterized in that the CpG site to be tested is located in a region near the 3 'end of the probe; preferably, the CpG site to be tested is located 3' near the probe One-third of the area;

   Preferably, the 5 'end of the probe is near the 3' end of the forward primer.
10. The method according to any one of claims 1-9, when multiple isolated CpG sites are detected at the same time, each CpG site contains a corresponding pair of primers and a pair of oligonuclei covering the CpG site to be tested Glycine probes, and the primer amplification regions of each CpG site to be tested do not overlap.
11. The method according to any one of claims 1 to 10, wherein in step c), the methylation state of the DNA to be tested is analyzed by a methylation ratio or a methylation percentage parameter PM;

    Preferably, the methylation ratio is methylation / (methylation + non-methylation)

    Preferably, the methylation percentage parameter PM is methylation / (methylation + non-methylation) × 100%;

    More preferably, the methylation percentage parameter PM = methylated fluorescence value / (methylated fluorescence value + unmethylated fluorescence value) × 100%;

    Still more preferably, the methylation percentage parameter PM = 100 / (1 + 1/2 ^-ΔCT ), ^ΔCT = CT _{methylated fluorescence—} CT _{unmethylated fluorescence} .
12. A reagent for detecting DNA methylation, characterized in that the reagent contains at least one pair of oligonucleotide probes, and the probes are probes connected to MGB;

    Preferably, the reagent does not contain a fully methylated standard;

    Preferably, one of the pair of oligonucleotide probes specifically binds a CG sequence and the other specifically binds a TG sequence;

    Preferably, the pair of oligonucleotide probes is a pair of Taqman probes. The 5 ′ end of each Taqman probe is connected to a fluorescent group, and the 3 ′ end is connected to a quencher and a -MGB group. The fluorophores attached to the 5 'end of each Taqman probe have different emission wavelengths;

    Preferably, for each Taqman probe, the fluorophore is selected from any one of FAM, VIC, ROX, TAMRA, SYTO9, JOE / TET / HEX, Texas Red, and NED / BODIPY / TMR-X;

    Preferably, the quencher is selected from one of NFQ, BHQ1 and BHQ2;

    Preferably, the length of the probe is between 10 and 20 bp; more preferably, the length of the probe is between 12 and 18 bp;

    Preferably, the CpG site to be tested is located in a region near the 3 'end of the probe; preferably, the CpG site is located in a third region near the 3' end of the probe.
13. The reagent according to claim 12, wherein the reagent further comprises at least a pair of primers; the pair of primers corresponds to a pair of probes;

    Preferably, the length of the primer amplification is 50-200bp;

    Preferably, the 5 'end of the probe is near the 3' end of the forward primer.
14. The reagent according to any one of claims 12-13, wherein the reagent further contains a transforming agent, and the transforming agent is selected from one of hydrazine, bisulfite, and bisulfite. Or several

    Preferably, the converting agent is selected from bisulfite.
15. The reagent according to any one of claims 12 to 14, wherein the reagent further comprises one or more of a DNA polymerase, dNTPs, Mg ²⁺ ions, and a buffer solution;

    Preferably, it contains DNA polymerase, dNTPs, Mg ²⁺ ions and a buffer.
16. The reagent according to any one of claims 12 to 15, characterized in that when there are a plurality of pairs of primers and a plurality of pairs of corresponding probes, the amplification regions of each pair of primers do not overlap.
17. A kit for detecting DNA methylation, characterized in that the kit contains the reagent according to any one of claims 12-16.
18. A system for detecting DNA methylation, characterized in that the system contains:

    a) DNA methylation detection building block, and,

    b) output components;

    The DNA methylation detecting member contains the reagent or kit according to any one of claims 12-17;

    Preferably, the output member is used to output a methylation ratio or a methylation percentage parameter PM.
19. Use of the method, reagent, kit, or system according to any one of claims 1 to 18 in the diagnosis of a disease;

    Preferably, the disease is a methylation-related disease;

    Or preferably, the disease is selected from the group consisting of: tumor; CNS dysfunction, injury; brain injury; psychiatric disorder; dementia; cardiovascular disease; gastrointestinal disease; respiratory disease; inflammation, infection, immunity; skin , Muscle, connective tissue or bone disorders; endocrine and metabolic disorders; headache or sexual dysfunction;

    More preferably, the disease is selected from a tumor.
20. The method, reagent, kit, or system according to any one of claims 1 to 18 for use in cell line authentication, tissue source identification, prenatal diagnosis, microbial identification, or species germline identification.
21. A method for diagnosing a disease, wherein the method uses the method, reagent, kit, or system according to any one of claims 1 to 18 to detect the DNA methylation status of a sample to be tested, and To determine whether the disease, disease probability, course, or type of disease; preferably, by comparing the methylation status of the sample to be tested with the normal sample, determine whether the disease, risk, probability, course, disease Types of;

    Preferably, the disease is a methylation-related disease;

    Or preferably, the disease is selected from the group consisting of: tumor; CNS dysfunction, injury; brain injury; psychiatric disorder; dementia; cardiovascular disease; gastrointestinal disease; respiratory disease; inflammation, infection, immunity; skin , Muscle, connective tissue or bone disorders; endocrine and metabolic disorders; headache or sexual dysfunction;

    More preferably, the disease is selected from a tumor.
22. A disease diagnosis system, comprising the system for detecting DNA methylation according to claim 18; preferably, the disease diagnosis system further comprises a result judgment component;

    More preferably, the result judgment component compares the methylation status of the test sample with the normal sample, and outputs whether or not the disease is present, the risk of the disease, and the probability of the disease according to the difference or difference between the sample to be tested and the normal sample. Course of disease, one or more of the types of disease;

    Preferably, the disease is a methylation-related disease;

    Or preferably, the disease is selected from the group consisting of: tumor; CNS dysfunction, injury; brain injury; psychiatric disorder; dementia; cardiovascular disease; gastrointestinal disease; respiratory disease; inflammation, infection, immunity; skin , Muscle, connective tissue or bone disorders; endocrine and metabolic disorders; headache or sexual dysfunction;

    More preferably, the disease is selected from a tumor.

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