CN114214397A

CN114214397A - Methylation detection method

Info

Publication number: CN114214397A
Application number: CN202111586076.3A
Authority: CN
Inventors: 赵辉; 贺綦; 高蕾
Original assignee: Ankai Life Technology Suzhou Co ltd
Current assignee: Ankai Life Technology Suzhou Co ltd
Priority date: 2021-12-20
Filing date: 2021-12-20
Publication date: 2022-03-22

Abstract

The present application relates to a method for methylation detection, comprising the steps of: obtaining PCR primers specific to a methylation site of a target gene, the PCR primers comprising an upstream primer and a downstream primer, wherein the 5' end of the upstream primer comprises an amino modification; and the 5' end of the downstream primer is provided with a label; coupling the upstream primer with a carrier to obtain a carrier-upstream primer conjugate; and carrying out PCR amplification on the sample by using the carrier-upstream primer conjugate and the downstream primer. The method is simple to operate, multiple in detection items and high in repeatability; can be applied to various sample types and multi-site DNA methylation detection in various fields.

Description

Methylation detection method

Technical Field

The application relates to the field of biomedicine, in particular to a methylation detection method.

Background

Currently, DNA methylation is the most studied epigenetic mechanism that plays an important role in maintaining normal cellular function, chromatin structure modification, X chromosome inactivation, genomic imprinting, embryonic development, and human tumorigenesis. In a healthy human genome, for example, CpG sites in CpG islands are usually unmethylated. When tumors occur, the non-methylation degree of CpG sequences except the CpG island of the cancer suppressor gene is increased, and the CpG in the CpG island is in a high methylation state.

Currently, there are several methods for studying DNA methylation levels:

methylation sensitive restriction enzyme (MS-RE) method, however, CG sequence recognition of the MSRE method has certain limitation, and false positive problem is caused when enzyme is not completely digested;

methylation-specific PCR (MS-PCR) is a common method for detecting gene methylation at present. However, the method has very high requirements on primer design, and the quality of primer design is a key factor of amplification success and failure.

Methylation sensitive high resolution melting curve analysis (MS-HRM) methods, however, require high instrumentation, require a fluorescent quantitative PCR instrument with an HRM module, and require the use of saturated fluorescent dyes in the real-time quantitative PCR process. Methylation detection by using the MS-HRM technology can only analyze the overall methylation condition of the detected fragments, and can not clearly determine the methylation state of each CpG site.

In combination with the Bisulfite Sequencing PCR (BSP) method, the method is not easy to be operated in large scale, and the process is complicated and expensive.

The matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MassARRAY) method, however, the instrument used in the method is expensive and is not favorable for clinical application.

Therefore, a method which is simple to operate and high in repeatability is urgently needed; can be applied to a plurality of sample types and a plurality of field multi-site DNA methylation detection methods.

Disclosure of Invention

The present application provides a method for methylation detection. Wherein the method may make DNA methylation detection more sensitive, accurate, increase throughput, and/or be more suitable for clinical use.

In one aspect, the present application provides a method of methylation detection, comprising the steps of: a) obtaining PCR primers specific to a methylation site of a target gene, the PCR primers comprising an upstream primer and a downstream primer, wherein the 5' end of the upstream primer comprises an amino modification; and the 5' end of the downstream primer is provided with a label; b) coupling the upstream primer with a carrier to obtain a carrier-upstream primer conjugate; and carrying out PCR amplification on the sample by using the carrier-upstream primer conjugate and the downstream primer.

In certain embodiments, the amino modification comprises having an amino modifying functional group at the 5' end of the forward primer, wherein the amino modifying functional group comprises at least one free amino group.

In certain embodiments, the Amino modifying functional group comprises 5' Amino C6-C18.

In certain embodiments, the forward primer comprises a forward specific sequence capable of specifically amplifying the methylation site of the gene of interest.

In certain embodiments, the amino modifying functional group is attached directly or indirectly to the 5' end of the upstream specific sequence.

In certain embodiments, the concentration of the forward primer is about 100 to about 300 nM.

In certain embodiments, the support comprises a solid phase support and/or a liquid phase support.

In certain embodiments, the support comprises a magnetic bead and/or a microsphere.

In certain embodiments, the surface of the support has hydroxyl modifications.

In certain embodiments, in the carrier-forward primer conjugate, the carrier and the forward primer are linked through the amino-modifying functional group.

In certain embodiments, in the carrier-forward primer conjugate, the carrier and the forward primer are linked through the interaction of an amino group in the amino-modified functional group and a hydroxyl group on the surface of the carrier.

In certain embodiments, in the vector-forward primer conjugate, the vector is attached to the 5' end of the forward primer.

In certain embodiments, the concentration of the carrier-forward primer conjugate is from about 1000 to about 10000 per reaction.

In certain embodiments, the downstream primer comprises a downstream specific sequence capable of specifically amplifying the methylation site of the gene of interest.

In certain embodiments, the label is capable of directly or indirectly visualizing the amplified product of the PCR amplification.

In certain embodiments, the label comprises a biotin label and/or a fluorescein label.

In certain embodiments, the label is linked directly or indirectly to the 5' end of the downstream specific sequence.

In certain embodiments, the concentration of the downstream primer is about 100 to about 300 nM.

In certain embodiments, the method further comprises the steps of: obtaining internal reference primers which are not specific to methylation sites of a target gene, wherein the internal reference primers comprise an internal reference upstream primer and an internal reference downstream primer; performing the PCR amplification of step b) by using the internal reference primer.

In certain embodiments, the 5' end of the reference primer forward primer comprises an amino modification.

In certain embodiments, the 5' end of the internal reference downstream primer carries a label.

In certain embodiments, the DNA in the sample is bisulfite treated and/or enzymatically converted.

In certain embodiments, the sample comprises genomic DNA, plasmid DNA, mitochondrial DNA, episomal DNA, and/or synthetic DNA.

In certain embodiments, the source of the sample comprises a cell, tissue, organ, and/or sample.

In certain embodiments, the cell, tissue, organ and/or sample is derived from a microorganism, plant, animal and/or human.

In certain embodiments, the sample is derived from a blood sample, a plasma sample, an FFPE sample, a tissue sample, a stool sample, and/or a urine sample.

In certain embodiments, the cell, tissue, organ and/or sample of the subject is derived from a tumor patient, and/or the cell, tissue comprises a tumor cell and/or a tumor tissue.

In certain embodiments, the tumor comprises a solid tumor and/or a non-solid tumor.

In certain embodiments, the vector is ligated to the amplification product of the PCR amplification of step b).

In certain embodiments, the PCR amplified product of step b) is labeled with a label that directly or indirectly allows for visualization of the amplified product.

In certain embodiments, the number of PCR amplifications described in step b) is 1.

In certain embodiments, the PCR amplification of step b) comprises a reaction step selected from the group consisting of: 1) pre-denaturation at 95 deg.C for 2-5min, thermal cycle at 95 deg.C for 15-20s, thermal cycle at 52-60 deg.C for 20-30s, 72 deg.C for 40s, 30-45 cycles, and final extension at 72 deg.C for 5-10 min; 2) pre-denaturation at 95 deg.C for 5-10min, thermal cycle at 95 deg.C for 15-30s, thermal cycle at 52-60 deg.C for 20-30s, 72 deg.C for 40s, 30-45 cycles, and final extension at 72 deg.C for 5-10 min; and, 3) pre-denaturation at 95 ℃ for 5-15min, thermal cycling at 95 ℃ for 15-30s, thermal cycling at 52-60 ℃ for 20-30s, thermal cycling at 72 ℃ for 40s, 30-45 cycles, and final extension at 72 ℃ for 5-10 min.

In certain embodiments, the method further comprises the steps of: c) generating a fluorescent signal from the PCR amplified product in the step b) and detecting, and obtaining the methylation modification condition of the sample according to the fluorescent signal.

In certain embodiments, the detecting comprises detecting using a liquid phase chip system.

In certain embodiments, the biotin can be combined with a streptavidin-labeled fluorescent dye in the amplification product to generate a fluorescent signal.

In certain embodiments, the fluorescent label is capable of generating a fluorescent signal in the amplification product.

In another aspect, the present application provides a methylation site upstream primer capable of specifically amplifying the gene of interest, the 5' end of which comprises an amino modification.

In certain embodiments, the upstream primer has an amino modifying functional group at the 5' end, wherein the amino modifying functional group comprises at least one free amino group.

In certain embodiments, the upstream specific sequence comprises a nucleotide sequence set forth in any one of SEQ ID nos. 1, 3-4.

In certain embodiments, the amino modifying functional group of the forward primer is directly or indirectly linked to the 5' end of the upstream specific sequence.

In another aspect, the present application provides a PCR primer pair capable of specifically amplifying a methylation site of the gene of interest, comprising an upstream primer and a downstream primer according to the present application.

In certain embodiments, the downstream specific sequence comprises a nucleotide sequence set forth in any one of SEQ ID nos. 2, 6-7.

In certain embodiments, the 5' end of the downstream primer carries a label.

In certain embodiments, the label comprises biotin and/or a fluorescent label.

In another aspect, the present application provides a methylation detection kit comprising an upstream primer described herein, and/or a PCR primer pair described herein.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The brief description of the drawings is as follows:

FIG. 1 shows a schematic flow chart of the coupling of the upstream primer to the magnetic bead according to the present application.

FIG. 2 is a schematic flow diagram of a method described herein.

FIG. 3 is a schematic flow diagram of a method described herein.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

Definition of terms

In the present application, the term "methylation" generally refers to the process of covalent bonding between the 5-carbon atom of cytosine on DNA and a methyl group, whereby cytosine is modified to 5-methylcytosine (5-methylcytosine, 5 mC). The types of DNA methylation can be divided into maintenance DNA methylation (maintence DNA methylation) and de novo methylation (de novo methylation). Maintaining methylation refers to the process of methylation modification at the corresponding position of the daughter strand during the process of half-retention replication of DNA by methyltransferase. The methylation process is a process of methylation on an original unmethylated DNA double strand by the action of methyltransferase, and then maintaining a stable DNA methylation state by the maintenance methylase. The methylation may also include the formation of N6-methylpurine (N6-mA) and 7-methylguanine (7-mG). Abnormal changes in methylation are prevalent in a variety of tumors, and abnormal DNA methylation status is one of the important features of tumors.

In the present application, the term "forward primer" is generally referred to as a sense strand (sense primer). The DNA molecule is a double strand in which the 5 '-3' strand is called the plus strand and the 3 '-5' strand is called the minus strand. The strand synthesized by the forward primer may have the same sequence as the plus strand. The strand synthesized by the forward primer may be complementary to the sequence of the minus strand.

In the present application, the term "reverse primer" generally refers to an antisense strand (antisense primer), and the strand synthesized by the reverse primer may be identical to the sequence of the minus strand. The strand synthesized by the reverse primer may be complementary to the sequence of the plus strand.

In the present application, the term "upstream specific sequence" generally refers to a nucleotide sequence of the upstream primer that can specifically amplify a methylation site of a gene of interest. For example, the upstream specific sequence may be identical to the sequence of the 5 '-3' strand of the DNA sequence in the vicinity of the methylation site of the target gene (i.e., the nucleotides 5 'upstream and 3' downstream of the methylation site). For example, the upstream specific sequence may be identical to a nucleotide sequence of at least 2 bases 5 'upstream and at least 2 bases 3' downstream of the methylation site in the 5 '-3' strand of the target gene.

In the present application, the term "downstream specific sequence" generally refers to a nucleotide sequence in the downstream primer that can specifically amplify a methylation site of a gene of interest. For example, the downstream specific sequence may be identical to the sequence of the 3 '-5' strand of the DNA sequence in the vicinity of the methylation site of the target gene (i.e., the nucleotides 5 'upstream and 3' downstream of the methylation site). For example, the upstream specific sequence may be identical to the sequence of nucleotides of at least 2 bases upstream and at least 2 bases downstream of the methylation site in the 3 '-5' strand of the target gene.

In the present application, the term "amino modification" generally refers to a modification such that it comprises an amino (e.g., free amino) functional group. For example, the amino group modification includes a modification such that an amino group at the N-terminus (e.g., an amino group free at the N-terminus) is produced.

In the present application, the term "label" generally refers to a marker that enables detection of PCR amplification products.

In this application, the term "liquid phase chip system" generally refers to a multifunctional suspended dot matrix instrument. The liquid phase chip system can be Multi-analysis substrate Array, MASA, flexible Multi-analysis Profiling or xMAP. The liquid phase chip system can be used for detecting biological macromolecules such as protein, nucleic acid and the like. The principle of the liquid phase chip system is that at least one probe suspended in the liquid phase system can generate different signals under the activation of laser after being combined with target molecules. The liquid chip system can detect only a small amount of samples and can perform qualitative and quantitative detection simultaneously.

In the present application, the term "PCR primer pair" generally refers to a pair of nucleotide sequences capable of efficiently amplifying a template DNA. For example, the PCR primer pair can include the forward primer and the reverse primer.

Detailed Description

Methylation detection method

According to the methylation detection method, the steps of specific amplification of the methylation sites of the target genes and hybridization of the amplification products and the carrier are realized through one round of PCR, so that the complicated steps of methylation detection are simplified, and the times of uncovering the sample involved in the detection process are reduced.

Specifically, in the present application, the forward primer may be coupled to a carrier (e.g., a magnetic bead and/or a microsphere) by including an amino modification at its 5' end, thereby obtaining a carrier-forward primer conjugate; the downstream primer may have a label (e.g., a biotin label or a fluorescent label) at its 5' end. The upstream and downstream primers can be used to perform PCR 1 round amplification on a sample (e.g., a sample that has been bisulfite treated). The amplification product obtained may comprise both the coupled carrier (e.g., magnetic beads and/or microspheres) and the biotin label or fluorescent label. For amplification products containing both coupled carriers (e.g., magnetic beads and/or microspheres) and fluorescent labels, the fluorescence signal can be directly detected, and methylation detection results can be obtained from the fluorescence signal; for amplification products that include both coupled supports (e.g., magnetic beads and/or microspheres) and biotin labels, Streptavidin (SAPE) can be used to generate a fluorescent signal, and methylation detection can be obtained from the fluorescent signal.

Upstream primer

In the present application, the amino modification may include providing the 5' end of the upstream primer with an amino modification functional group, wherein the amino modification functional group comprises at least one free amino group.

In the present application, the Amino modifying functional group may comprise 5' Amino C6-C18. For example, the amino acid modification functional group may comprise a 5 'amino C6 modification and/or a 5' amino C12 modification. For example, the Amino modifying functional group may comprise Amino Modifier C12. The Amino Modifier C12 may have the following structure:

in the present application, the upstream primer may comprise an upstream specific sequence capable of specifically amplifying the methylation site of the gene of interest. For example, the upstream specific sequence may be identical to at least a portion of the sequence of the strand comprising 5 '-3' of the DNA sequence capable of specifically amplifying the methylation site of the gene of interest.

In the present application, the amino modifying functional group may be attached directly or indirectly to the 5' end of the upstream specific sequence. For example, the amino acid modifying functional group may be attached directly to the 5' end of the upstream specific sequence, or may be attached through one or more nucleotides (e.g., these nucleotides serve as a linker sequence).

In the present application, the concentration of the forward primer may be from about 100 to about 300 nM. For example, it may be about 100 to about 280nM, about 100 to about 260nM, about 100 to about 240nM, about 100 to about 220nM, about 100 to about 200nM, about 100 to about 180nM, about 100 to about 160nM, about 100 to about 140nM, about 120 to about 300nM, about 160 to about 300nM, or about 200 to about 300 nM.

Magnetic beads and conjugates

In the present application, the support may comprise a solid phase support and/or a liquid phase support.

In the present application, the solid support may comprise a chip.

In the present application, the liquid phase carrier may include magnetic beads and/or microspheres. The magnetic beads and/or microspheres may have a silicon-based material. The magnetic beads and/or microspheres may have a carboxyl or amino modification. The magnetic beads and/or microspheres may be selected from the products of Miltenyi, usa, Dynabeads, magseroon beads, germany, Ademtech, france. The magnetic beads and/or microspheres may have a particle size of about 1-5 μm.

In the present application, the surface of the support may have a hydroxyl modification.

In the present application, in the carrier-forward primer conjugate, the carrier and the forward primer may be linked through the amino-modified functional group. For example, in the carrier-forward primer conjugate, the carrier and the forward primer can be connected through the interaction of the amino group in the amino group modification functional group and the hydroxyl group on the surface of the carrier.

In the present application, in the carrier-forward primer conjugate, the carrier may be linked to the 5' end of the forward primer.

In the present application, the concentration of the carrier-upstream primer conjugate may be from about 1000 to about 10000 per reaction. For example, the concentration of the carrier-upstream primer conjugate can be from about 2500 to about 10000 per reaction.

Downstream primer

In the present application, the downstream primer may comprise a downstream specific sequence capable of specifically amplifying the methylation site of the gene of interest. For example, the downstream specific sequence may be identical to at least a portion of the sequence of the strand comprising the 3 '-5' of the DNA sequence capable of specifically amplifying the methylation site of the gene of interest.

In the present application, the label may directly or indirectly color the PCR amplification product.

In the present application, the label may comprise a biotin label and/or a fluorescein label. For example, the fluorescein label can include CY3, HEX, BODIPY-TMRX, and/or TAMRA. For example, the fluorescein label can be BODIPY-TMRX.

In the present application, the label may be linked directly or indirectly to the 5' end of the downstream specific sequence.

In the present application, the concentration of the downstream primer may be about 100 to about 300 nM. For example, it may be about 100 to about 280nM, about 100 to about 260nM, about 100 to about 240nM, about 100 to about 220nM, about 100 to about 200nM, about 100 to about 180nM, about 100 to about 160nM, about 100 to about 140nM, about 120 to about 300nM, about 160 to about 300nM, or about 200 to about 300 nM.

Sample(s)

In the present application, the DNA in the sample may be converted by bisulfite treatment or enzymatic conversion. The method can convert all unmethylated cytosines to uracils, which can be converted to thymines in a subsequent PCR amplification. Whereas methylated cytosines remain unchanged during this process. The bisulfite treatment or enzymatic conversion may be accomplished using a kit.

In the present application, the sample may include genomic DNA, plasmid DNA, mitochondrial DNA, episomal DNA, and/or synthetic DNA.

In the present application, the source of the sample may comprise a cell, a tissue, an organ and/or a sample. For example, the cells, tissues, organs and/or samples may be of microbial, plant, animal and/or human origin. For example, the sample may be derived from a blood sample, a plasma sample, an FFPE sample, a tissue sample, a stool sample, and/or a urine sample. For example, the cell, tissue, organ and/or sample of the subject may be derived from a tumor patient, and/or the cell, tissue may comprise a tumor cell and/or a tumor tissue. For example, the tumor may comprise a solid tumor and/or a non-solid tumor. The methods of the present application may be adapted for use with different patients for indications and/or different sources of samples obtained from the patients.

Method

In this application, the detection method may further include the steps of: the sample DNA was treated with bisulfite.

In the present application, a methylation-specific product comprising the vector and a fluorescent label can be obtained via step b); for example, methylation specific products with magnetic beads and/or microsphere-fluorescent labels can be obtained. For example, methylation specific products with magnetic beads and/or microsphere-biotin labels can be obtained.

In the present application, the amplification product of the PCR amplification of step b) may be linked to the vector.

In the present application, the amplification product of the PCR amplification of step b) may carry a label capable of directly or indirectly developing the color of the amplification product.

In the present application, the number of times of PCR amplification in step b) may be 1.

In the present application, the PCR amplification of step b) may comprise a reaction step selected from the group consisting of: 1) pre-denaturation at 95 deg.C for 2-5min, thermal cycle at 95 deg.C for 15-20s, thermal cycle at 52-60 deg.C for 20-30s, 72 deg.C for 40s, 30-45 cycles, and final extension at 72 deg.C for 5-10 min; 2) pre-denaturation at 95 deg.C for 5-10min, thermal cycle at 95 deg.C for 15-30s, thermal cycle at 52-60 deg.C for 20-30s, 72 deg.C for 40s, 30-45 cycles, and final extension at 72 deg.C for 5-10 min; and, 3) pre-denaturation at 95 ℃ for 5-15min, thermal cycling at 95 ℃ for 15-30s, thermal cycling at 52-60 ℃ for 20-30s, thermal cycling at 72 ℃ for 40s, 30-45 cycles, and final extension at 72 ℃ for 5-10 min.

For example, the PCR amplification may comprise the following reaction steps: pre-denaturation at 95 deg.C for 2-5min, thermal cycle at 95 deg.C for 15-20s, thermal cycle at 52-60 deg.C for 20-30s, 72 deg.C for 40s, 30-45 cycles, and final extension at 72 deg.C for 5-10 min. For example, the PCR amplification may comprise the following reaction steps: pre-denaturation at 95 deg.C for 5-15min, thermal cycle at 95 deg.C for 15-30s, thermal cycle at 52-60 deg.C for 20-30s, 72 deg.C for 40s, 30-45 cycles, and final extension at 72 deg.C for 5-10 min.

In the present application, the method may further comprise the steps of: obtaining internal reference primers which are not specific to methylation sites of a target gene, wherein the internal reference primers comprise an internal reference upstream primer and an internal reference downstream primer; performing the PCR amplification of step b) by using the internal reference primer.

In the present application, the 5' end of the forward primer of the reference primer may include an amino modification. In the present application, the 5' end of the internal reference downstream primer may carry a label.

In the present application, the reference primer can be used as a control primer, which can not specifically amplify the methylation site of the target gene, but can amplify other reference genes (such as beta-actin gene), and thus can be used for detecting the accuracy of the PCR amplification system.

In the present application, the method may further comprise the steps of: c) generating a fluorescent signal from the PCR amplified product in the step b) and detecting, and obtaining the methylation modification condition of the sample according to the fluorescent signal.

In the present application, the detection may include detection using a liquid phase chip system.

In the present application, the biotin may be combined with a streptavidin-labeled fluorescent dye in the amplification product to generate a fluorescent signal.

In the present application, the fluorescent label may generate a fluorescent signal in the amplification product.

In the present application, a biotin-streptavidin (or avidin) system may be used to cause the amplification product to generate a fluorescent signal. For example, the beads and/or microspheres may be coated with streptavidin, and the beads and/or microspheres may be coated with streptavidin

The amplification products of biotin bind to produce a fluorescent signal. In some cases, instead of the biotin-chain enzyme avidin (or avidin) system, other different affinity binding partners may be used, e.g. antigens/haptens and antibodies, or enzymes and corresponding substrates may be used.

In the present application, step c) may comprise detecting the fluorescent signal generated by the methylation specific product comprising the support and the fluorescent label (e.g.methylation specific product with magnetic beads and/or microsphere-fluorescent label) and performing a data analysis.

In the present application, step c) may comprise detecting the fluorescent signal generated by the methylation specific product comprising the support and the fluorescent label (e.g.methylation specific product with a magnetic bead and/or a microsphere-biotin label) and performing a data analysis. For example, the methylation specific products with magnetic beads and/or microsphere-biotin labels can be fluorescently labeled using streptavidin-labeled fluorescent dye (SAPE). In the present application, the reaction conditions for the fluorescent label may be about 25 to about 37 ℃ for about 10 to about 30 min.

In the present application, the detection may be detected by a liquid phase chip system.

The methods described herein may have one or more of the following advantages:

1. the method is simple to operate, saves a large amount of detection time, and can carry out detection quickly and in high flux (for example, more than 100 samples can be detected for 1 time).

2. And the PCR product can be detected on the machine after being amplified, so that the risk of uncovering pollution of the PCR product is reduced.

3. The detection accuracy is high, the specificity is strong, the amount of the needed DNA sample is small, and the method is suitable for detecting trace DNA samples.

4. Multiple assays can be performed.

5. Can be applied to the multi-site DNA methylation detection in various fields, such as cancer screening, disease diagnosis, scientific research and the like.

6. Can be used for a variety of sample types (e.g., human blood samples, plasma samples, FFPE samples, tissue samples, stool samples, urine samples, and plant microorganisms).

In the present application, the 5' end of the upstream primer has an amino-modifying functional group, wherein the amino-modifying functional group comprises at least one free amino group.

In the present application, the Amino modifying functional group comprises 5' Amino C6-C18.

In the present application, the upstream primer comprises an upstream specific sequence capable of specifically amplifying the methylation site of the gene of interest.

In the present application, the upstream specific sequence comprises a nucleotide sequence shown in any one of SEQ ID NO.1, 3-4.

In the present application, the upstream primer has the amino-modifying functional group directly or indirectly linked to the 5' end of the upstream specific sequence.

In the present application, the downstream primer comprises a downstream specific sequence capable of specifically amplifying the methylation site of the gene of interest.

In the present application, the downstream specificity sequence comprises a nucleotide sequence shown in any one of SEQ ID NO.2, 6-7.

In the present application, the 5' end of the downstream primer carries a label.

In the present application, the label is capable of directly or indirectly developing the amplified product of the PCR amplification.

In the present application, the label comprises biotin and/or a fluorescent label.

In the present application, the label is linked directly or indirectly to the 5' end of the downstream specific sequence.

The present application also provides the following embodiments:

1. a method of methylation detection, comprising the steps of:

a) obtaining PCR primers specific to a methylation site of a target gene, the PCR primers comprising an upstream primer and a downstream primer, wherein the 5' end of the upstream primer comprises an amino modification; and the 5' end of the downstream primer is provided with a label;

b) coupling the upstream primer with a carrier to obtain a carrier-upstream primer conjugate; and carrying out PCR amplification on the sample by using the carrier-upstream primer conjugate and the downstream primer.

2. The method of embodiment 1, wherein the amino modification comprises having an amino modifying functional group at the 5' end of the forward primer, wherein the amino modifying functional group comprises at least one free amino group.

3. The method of embodiment 2, wherein the Amino modifying functional group comprises 5' Amino C6-C18.

4. The method according to any one of embodiments 1-3, wherein the upstream primer comprises an upstream specific sequence capable of specifically amplifying the methylation site of the gene of interest.

5. The method of embodiment 4, wherein the amino modifying functional group is attached directly or indirectly to the 5' end of the upstream specific sequence.

6. The method according to any one of embodiments 1-5, wherein the concentration of the forward primer is about 100 to about 300 nM.

7. The method of any one of embodiments 1-6, wherein the support comprises a solid phase support and/or a liquid phase support.

8. The method of any one of embodiments 1-7, wherein the support comprises magnetic beads and/or microspheres.

9. The method of any one of embodiments 1-8, wherein the surface of the support has a hydroxyl modification.

10. The method according to any one of embodiments 2-9, wherein in the carrier-forward primer conjugate, the carrier and the forward primer are linked through the amino-modifying functional group.

11. The method according to any one of embodiments 2-10, wherein in the carrier-forward primer conjugate, the carrier and the forward primer are linked by interaction of an amino group in the amino-modified functional group with a hydroxyl group on the surface of the carrier.

12. The method of any one of embodiments 1-11, wherein in the carrier-forward primer conjugate, the carrier is attached to the 5' end of the forward primer.

13. The method according to any one of embodiments 1-12, wherein the concentration of the carrier-upstream primer conjugate is from about 1000 to about 10000 per reaction.

14. The method according to any one of embodiments 1-13, wherein the downstream primer comprises a downstream specific sequence capable of specifically amplifying the methylation site of the gene of interest.

15. The method according to any one of embodiments 1-14, wherein the label is capable of directly or indirectly visualizing an amplification product of the PCR amplification.

16. The method of any one of embodiments 1-15, wherein the label comprises a biotin label and/or a fluorescein label.

17. The method according to any one of embodiments 1-16, wherein the label is linked directly or indirectly to the 5' end of the downstream specific sequence.

18. The method of any one of embodiments 1-17, wherein the concentration of the downstream primer is about 100 to about 300 nM.

19. The method according to any one of embodiments 1-18, wherein the method further comprises the steps of: obtaining internal reference primers which are not specific to methylation sites of a target gene, wherein the internal reference primers comprise an internal reference upstream primer and an internal reference downstream primer; performing the PCR amplification of step b) by using the internal reference primer.

20. The method of embodiment 19, wherein the 5' end of the internal reference primer forward primer comprises an amino modification.

21. The method of any one of embodiments 19-20, wherein the 5' end of the internal reference downstream primer carries a label.

22. The method according to any one of embodiments 1-21, wherein the DNA in the sample is bisulfite treated and/or enzymatically converted.

23. The method of any one of embodiments 1-22, wherein the sample comprises genomic DNA, plasmid DNA, mitochondrial DNA, episomal DNA, and/or synthetic DNA.

24. The method of any one of embodiments 1-23, wherein the source of the sample comprises a cell, a tissue, an organ, and/or a sample.

25. The method of embodiment 24, wherein the cell, tissue, organ and/or sample is derived from a microorganism, plant, animal and/or human.

26. The method of any one of embodiments 1-25, wherein the sample is derived from a blood sample, a plasma sample, an FFPE sample, a tissue sample, a stool sample, and/or a urine sample.

27. The method according to any one of embodiments 24-26, wherein the cell, tissue, organ and/or sample of the subject is derived from a tumor patient, and/or the cell, tissue comprises a tumor cell and/or a tumor tissue.

28. The method of embodiment 27, wherein the tumor comprises a solid tumor and/or a non-solid tumor.

29. The method according to any one of embodiments 1-28, wherein the amplification product of the PCR amplification of step b) is linked to the vector.

30. The method according to any one of embodiments 1-29, wherein the amplification product of the PCR amplification of step b) carries a label capable of directly or indirectly developing the color of the amplification product.

31. The method according to any one of embodiments 1-30, wherein the number of PCR amplifications of step b) is 1.

32. The method according to any one of embodiments 1-31, wherein the PCR amplification of step b) comprises a reaction step selected from the group consisting of:

1) pre-denaturation at 95 deg.C for 2-5min, thermal cycle at 95 deg.C for 15-20s, thermal cycle at 52-60 deg.C for 20-30s, 72 deg.C for 40s, 30-45 cycles, and final extension at 72 deg.C for 5-10 min;

2) pre-denaturation at 95 deg.C for 5-10min, thermal cycle at 95 deg.C for 15-30s, thermal cycle at 52-60 deg.C for 20-30s, 72 deg.C for 40s, 30-45 cycles, and final extension at 72 deg.C for 5-10 min; and the number of the first and second groups,

3) pre-denaturation at 95 deg.C for 5-15min, thermal cycle at 95 deg.C for 15-30s, thermal cycle at 52-60 deg.C for 20-30s, 72 deg.C for 40s, 30-45 cycles, and final extension at 72 deg.C for 5-10 min.

33. The method of any one of embodiments 1-32, further comprising the steps of:

c) generating a fluorescent signal from the PCR amplified product in the step b) and detecting, and obtaining the methylation modification condition of the sample according to the fluorescent signal.

34. The method of embodiment 33, wherein said detecting comprises detecting using a liquid phase chip system.

35. The method according to any one of embodiments 33-34, wherein the biotin is capable of binding to a streptavidin-labeled fluorescent dye in the amplification product to generate a fluorescent signal.

36. The method of any one of embodiments 33-35, wherein the fluorescent label is capable of generating a fluorescent signal in the amplification product.

37. And the 5' end of the methylation site upstream primer can specifically amplify the target gene and comprises amino modification.

38. The forward primer of embodiment 37, wherein the 5' end of the forward primer has an amino modifying functional group, wherein the amino modifying functional group comprises at least one free amino group.

39. The forward primer of embodiment 38 wherein the Amino modifying functional group comprises 5' Amino C6-C18.

40. The forward primer of any one of embodiments 37-39 comprising a forward specific sequence capable of specifically amplifying a methylation site of the gene of interest.

41. The upstream primer according to embodiment 40, wherein the upstream specific sequence comprises a nucleotide sequence set forth in any one of SEQ ID No.1, 3-4.

42. The forward primer of any of embodiments 37-41 wherein the amino modifying functional group is attached directly or indirectly to the 5' end of the upstream specific sequence.

43. A PCR primer pair capable of specifically amplifying a methylation site of the gene of interest, comprising an upstream primer and a downstream primer according to any one of embodiments 1-36.

44. The PCR primer pair of embodiment 43, wherein the downstream primer comprises a downstream specific sequence capable of specifically amplifying the methylation site of the gene of interest.

45. The PCR primer pair according to embodiment 44, wherein the downstream specific sequence comprises a nucleotide sequence set forth in any one of SEQ ID No.2, 6-7.

46. The PCR primer pair according to any one of embodiments 43-45, wherein the 5' end of the downstream primer carries a label.

47. The PCR primer pair according to embodiment 46, wherein the label is capable of directly or indirectly visualizing an amplification product of the PCR amplification.

48. The PCR primer pair according to any one of embodiments 46-47, wherein the label comprises biotin and/or a fluorescent label.

49. The PCR primer pair according to any one of embodiments 46-48, wherein the label is directly or indirectly linked to the 5' end of the downstream specific sequence.

50. A methylation detection kit comprising a forward primer of any one of embodiments 37-42, and/or a PCR primer pair of any one of embodiments 43-49.

Without wishing to be bound by any theory, the following examples are intended only to illustrate the methods, uses, etc. of the present application and are not intended to limit the scope of the invention of the present application.

Examples

Example 1 magnetic bead primer coupling

1. The primers described in this example were synthesized by Kinsley Biotech, the reagent MES (2- (N-morpholine) ethanesulfonic acid) from Sigma Aldrich trade company, Sigma M3671, and EDAC N' - (ethyliminomethylene) -N, N-dimethyl-1, 3-propanediamine monohydrochloride from Sigma Aldrich trade company, Sigma E7750. Tween-20 (Tween-20) was obtained from Solambio T8220, and SDS (sodium dodecyl sulfate) was obtained from Solambio S8010.

2. The PCR upstream and downstream primers are designed aiming at a target gene methylation site, and the 5' end of the upstream primer is modified by Amino Modifier C12, so that the aim of coupling the probe Amino group with the magnetic bead hydroxyl group is fulfilled.

The primer sequence length is 18-25bp, the TM value is 55-65 DEG C

The sequence of the upstream primer is as follows: STEP 9-M-F5 'Amino Modifier C12-TTTTCGTTATGGTTCGGTTT-3' (SEQ ID NO.1)

The probe was diluted to 0.1nanomole (nanomole) with pure water for use.

3. The magnetic beads are MagPlex magnetic beads modified by surface hydroxyl of Luminex company, and are suitable for MAGOX or Luminex100/200 liquid phase suspension chip systems of the Luminex company. Other hydroxyl surface modified magnetic beads and other liquid phase chip systems can also be adopted, and are not limited herein.

4. A coupling step:

1) uncoupled MagPlex beads 1.25X 10⁶Adding into 1.5ml centrifuge tube, placing in magnetic rack for magnetic attraction for 2min, and removing supernatant.

2) Add 10. mu.L of 0.1M MES, pH 4.5, mix well by shaking, sonicate for 20 s.

3) Add 2. mu.L of probe (0.1nanomole) to the tube and vortex to mix.

4) Preparing 10mg/ml EDAC, adding 0.75 μ L into the centrifuge tube, mixing by vortex, and standing in dark for 30 min.

5) Adding freshly prepared EDAC of 10mg/ml, adding 0.75 mu L into the centrifuge tube, mixing uniformly by vortex, and standing for 30min in the dark.

6) 1ml of Tween-20 with the volume percentage of 0.02 percent is added into the centrifuge tube, the centrifuge tube is placed in a magnetic rack for magnetic attraction for 2min, and the supernatant is sucked and discarded.

7) Adding 1ml SDS solution with volume percentage of 0.1%, placing in a magnetic rack for magnetic attraction for 2min, and absorbing and removing the supernatant.

8) Adding 50. mu.L of TE solution (the substance composition is 10mM Tris-HCl and 1mM EDTA) with pH8.0, and vortexing and mixing to obtain the coupled capture magnetic bead conjugate.

9) Counting the number of the magnetic beads by using a hemocytometer, and if the magnetic beads need to be stored, storing the magnetic beads in a dark place at the temperature of 2-8 ℃.

Example 2 assay specificity of detection

1. The primers described in this example were synthesized by Kinry Biotech, and the plasmid containing the target region of M-STEP9, M-VIM, M-FOXE1, β -ACTIN methylation site and NC (region not containing the target region of methylation site) was synthesized by Kinry Biotech, Inc.; 2 × EpiArt HS Taq Master Mix was purchased from Nanjing Novowed Biotech GmbH (Vazyme, EM 202-01); TMAC (tetramethylammonium chloride solution) was purchased from Sigma aldrich trade ltd (Sigma T3411); sarkosyl solution (sodium lauryl sarcosinate) was purchased from Sigma aldrich trade ltd (Sigma L7414); Tris-HCl Tris (hydroxymethyl) aminomethane hydrochloride was purchased from Solambio T8230, Beijing Sorley technologies, Inc.; triton X-100 (polyethylene glycol p-isooctylphenyl ether) was purchased from Solambio T8200, Kyoto Soilebao Tech., Ltd.; EDTA (ethylene diamine tetraacetic acid) was purchased from Seiko Biotech, Inc. (BioFroxx 1340GR 500); tris (Tris hydroxymethyl aminomethane) was purchased from Seitland Biotechnology GmbH (BioFroxx 1115GR 500); NaCl (sodium chloride) was purchased from Tianda chemical reagent.

2. Primer probe design and synthesis

The PCR downstream primer is designed aiming at methylation sites, the purity of the primer reaches electrophoresis level (PAGE) or HPLC level, and biotin modification is carried out at the 5' end of the downstream primer

Primers were synthesized with the following sequences:

a downstream primer: STEP 9-M-R5 'BODIPY-TMRX-CTAAAAAACAATCCTAAACACAC-3' (SEQ ID NO.2)

Wherein BODIPY-TMRX represents a fluorescent modification.

The dry primers were dissolved by adding TE pH8.0 separately.

The preparation of the magnetic bead primer is carried out according to the condition that the quantity of the magnetic bead-primer conjugates is 250 per reaction and the concentration range of the downstream primer is 100 and 300 nM.

3. Synthesis of standards and negative controls

Respectively dissolving target region standard substance of M-STEP9, M-VIM, M-FOXE1, M-beta-ACTIN methylation sites and NC negative control standard substance dry powder in water without enzyme, and diluting to 10%¹⁵Copy/ml, 10-fold gradient dilution standard, 10 of each¹⁰Copy/ml as standard template, using M-VIM, M-STEP9, M-FOXE1, M- β -ACTIN to detect the specificity of STEP9 magnetic bead-primer conjugates and STEP9 primer, respectively.

4. Specificity analysis

1) And (3) diluting a target region standard substance of M-STEP9, M-VIM, M-FOXE1, M-beta-ACTIN methylation sites and an NC negative standard substance according to the requirements to serve as a template to be detected, and detecting according to the following STEPs.

2) The PCR amplification system of this example is shown in Table 1:

TABLE 1. 10. mu.l PCR amplification System for the specific analysis of this example

Composition (I)	Volume of
		Magnetic bead PCR primer mixture	2.8μl
2×EpiArt HS Taq Master Mix	5μl
		DNA template after transformation	2μl
ddH₂O	To 10. mu.l

After mixing, shaking uniformly, centrifuging instantaneously, and placing on a Bio-rad T100 for PCR reaction, wherein the reaction program is as follows:

3) washing: the PCR product of step 2) was removed from the PCR machine and immediately placed on a magnetic plate (V & P Scientific, VP771LD-4CS) for 60s before the supernatant was discarded. Adding 50 μ l of 1 XTM hybridization solution into each reaction well, shaking and mixing for 20s, placing on a magnetic plate for 30-60s, discarding the supernatant, and repeating for 1 time. Finally, 75. mu.l of 1 XTM hybridization solution was added to each reaction well, and the mixture was shaken and mixed for 20 seconds.

4) Liquid phase chip platform detection: detecting the product obtained in the step 3) by using a Luminex200 liquid phase chip platform, and operating the detection operation steps and parameter setting according to Luminex200 operation instructions.

5) And (3) judging a detection result: and comparing the detection result of the sample with the result of the negative standard, if the detection value of a certain gene is more than or equal to three times of the value of the negative standard, judging that the gene is positive, and if the detection value of a certain gene of the sample is less than three times of the value of the negative standard, judging that the gene is negative.

6) The detection results are shown in Table 2, wherein M-STEP9, M-VIM, M-FOXE1 and M-beta-ACTIN are target region standard products of methylation sites, and NC is negative standard product.

TABLE 2 detection results of magnetic bead-primer specificity

Position of	Sample(s)	B39_STEP9
			1(1,A1)	M-VIM	29
2(1,B1)	M-FOXE1	27
			3(1,C1)	M-β-ACTIN	19
4(1,D1)	M-STEP9	619
			5(1,E1)	NC	40

The detection method has the advantages that the STEP9 primers can specifically distinguish the M-STEP9 standard substance, and the specificity of the method is better.

Example 3 multiplex assay

1. The primers and probes described in this example were synthesized by the biosciences of Kinsley; the negative standard was bisulfite-converted human unmethylated DNA purchased from Zymo RESEARCH Bio (ZYMO RESEARCH, D5014); tissue extraction kit was purchased from blood/cell/tissue genomic DNA extraction kit (TIANGEN, DP304) produced by TIANGEN biochemical technology (beijing) ltd; 2 × EpiArt HS Taq Master Mix was purchased from Nanjing Novowed Biotech GmbH (Vazyme, EM 202-01); TMAC (tetramethylammonium chloride solution) was purchased from Sigma aldrich trade ltd (Sigma T3411); sarkosyl solution (sodium lauryl sarcosinate) was purchased from Sigma aldrich trade ltd (Sigma L7414); Tris-HCl Tris (hydroxymethyl) aminomethane hydrochloride, available from Solambio T8230, Inc., Beijing Sorley technologies, Inc.; triton X-100 (polyethylene glycol p-isooctylphenyl ether) was purchased from Solambio T8200, Kyoto Soilebao Tech., Ltd.; EDTA (ethylene diamine tetraacetic acid) was purchased from Seiko Biotech, Inc. (BioFroxx 1340GR 500); tris (Tris hydroxymethyl aminomethane) was purchased from Seitland Biotechnology GmbH (BioFroxx 1115GR 500); NaCl (sodium chloride) was purchased from Tianda chemical reagent.

2. Probe design and synthesis were performed according to the method described in example 1, with the following sequences

An upstream primer: FOXE 1-M-F5 'Amino Modifier C12-TTTGTTCGTTTTTCGATTGTTC-3' (SEQ ID NO.3)

An upstream primer: VIM-M-F5 'Amino Modifier C12-ATGTTACGCGTTTTTTTGTC-3' (SEQ ID NO.4)

An upstream primer: beta-ACTIN-M-F5 'Amino Modifier C12-TTTAATGTTACGTACGATTTTTC-3' (SEQ ID NO.5)

The bead-primer coupling was performed as described in example 1, and the coupled bead-primer conjugates were subjected to a cytometer to count the number of beads.

3. Primer design and synthesis were performed according to the method described in example 2, with the sequences as follows

Downstream primer FOXE 1-M-R5 'BODIPY-TMRX-TAACGCTATAAAACTCCTACCGC-3' (SEQ ID NO.6)

Downstream primer VIM-M-R5 'BODIPY-TMRX-AACTCCACCTTCTCGTTAATAC-3' (SEQ ID NO.7)

Internal reference gene primers:

downstream primer beta-ACTIN-M-R5 'BODIPY-TMRX-GTAGGATGGTATGGGGGA-3' (SEQ ID NO.8)

Wherein BODIPY-TMRX represents a fluorescent modification.

The dry primers were dissolved by adding TE pH8.0 separately.

The bead primer formulation was performed at 2500 per reaction per bead-primer conjugate, with each downstream primer concentration ranging from 100 and 300 nM.

4. Tissue sample extraction: DNA extraction was performed on 5 human colorectal cancer tissue samples and 5 non-colorectal cancer tissue samples using a blood/cell/tissue genome DNA extraction kit (DP304) manufactured by Tiangen Biochemical technology (Beijing) Ltd according to the method of the specification.

5. Human methylated DNA and human unmethylated DNA (ZYMO RESEARCH, D5014) after bisulfite conversion were used as positive and negative standards, and human tissue DNA samples after bisulfite conversion were used as samples to be tested.

6. Multiplex assays

1) The PCR amplification system of this example is shown in Table 3 below:

TABLE 3 10. mu.l PCR amplification System for multiplex detection in this example

Composition (I)	Volume of
		PCR primer mixture (2.5. mu.M)	2.8μl
2×EpiArt HS Taq Master Mix	5μl
		DNA template after transformation	2μl
ddH₂O	To 10. mu.l

2) washing: after the reaction, the 96-well plate was immediately placed on a magnetic plate for 60 seconds, and the supernatant was discarded. Adding 50 μ l of 1 XTM hybridization solution into each reaction well, shaking and mixing for 20s, placing on a magnetic plate for 30-60s, discarding the supernatant, and repeating for 1 time. Finally, 75. mu.l of 1 XTM hybridization solution was added to each reaction well, and the mixture was shaken and mixed for 20 seconds.

3) Liquid phase chip platform detection: detecting the product obtained in the step 2) by using a Luminex200 liquid phase chip platform, and operating the detection operation steps and parameter setting according to Luminex200 operation instructions.

4) And (3) judging a detection result: and comparing the detection result of the sample with the result of the negative standard, if the detection value of a certain gene is more than or equal to three times of the value of the negative standard, judging that the gene is positive, and if the detection value of a certain gene of the sample is less than three times of the value of the negative standard, judging that the gene is negative.

7. The results are shown in Table 4, with NC as a negative control and PC as a positive control.

TABLE 4 multiple assay results for colorectal cancer samples in this example

Position of	Sample(s)	B39_STEP9	B55_FOXE1	B66_VIM	B72_β-ACTIN
						1(1，A1)	C1	796	560	434	567
2(1，B1)	C2	902	784.5	64.5	795
						3(1，C1)	C3	754	56	897.5	340
4(1，D1)	C4	809	204.5	832	314.5
						5(1，E1)	C5	879.5	822	364	592
6(1，F1)	H1	55	43	43	551
						7(1，F1)	H2	35	90.5	54.5	657.5
8(1，G1)	H3	31	131	41	447
						9(1，A2)	H4	33.5	92.5	42	650
10(1，B2)	H5	32	59.5	52	542
						11(1，C2)	PC	847	652.5	548.5	632
12(1，D2)	NC	34.5	54	31	52
						13(1，E2)	H	41	43	52	37

The detection results of several positive control sites are all more than 3 times of the corresponding NC values, which proves that the method can simultaneously detect a plurality of sites; at least 2 sites of all 5 patient samples are positive, and all the controls are negative, so that the colorectal cancer samples can be well distinguished by the method.

Example 4 assay specificity of detection

1. The primers of this example were synthesized by Kinry Biotech, and the plasmid containing M-STEP9, M-VIM, M-FOXE1, the target region of the β -ACTIN methylation site and NC (the target region not containing the methylation site) was synthesized by Kinry Biotech, Inc.; 2 × Taq Master mix (required Taq enzyme, dNTP mix, MgCl2 and reaction buffer were prepared as a 2-fold concentration mixture) the required reagents were purchased from TAKALA (TAKALA, RR 001B); the SAPE stock solution was purchased from Life Technologies, S-866, seimei feishale Technologies (china); TMAC (tetramethylammonium chloride solution) was purchased from sigma aldrich trade ltd (SigmaT 3411); sarkosyl solution (sodium lauryl sarcosine solution) was purchased from sigma aldrich trade ltd (SigmaL 7414); Tris-HCl Tris (hydroxymethyl) aminomethane hydrochloride was purchased from Solambio T8230, Beijing Sorley technologies, Inc.; triton X-100 (polyethylene glycol p-isooctylphenyl ether) was purchased from SolambioT 8200, Soilebao technologies, Inc. of Beijing; EDTA (ethylene diamine tetraacetic acid) was purchased from Seiko Biotech, Inc. (BioFroxx 1340GR 500); tris (Tris hydroxymethyl aminomethane) was purchased from Seitland Biotechnology GmbH (BioFroxx 1115GR 500); NaCl (sodium chloride) was purchased from Tianda chemical reagent.

2. Primer probe design and synthesis

The PCR downstream primer is designed aiming at methylation sites, the purity of the primer is required to reach electrophoresis level (PAGE) or HPLC level, and the 5' end of the downstream primer is modified by biotin.

Primers were synthesized with the following sequences:

a downstream primer: STEP 9-M-R5 'Biotin-CTAAAAAACAATCCTAAACACAC-3'

Where Biotin represents the Biotin modification.

The dry primers were dissolved by adding TE pH8.0 separately.

The preparation of the magnetic bead primer was carried out according to the amount of the magnetic bead-primer conjugates being 2500/reaction and the downstream primer concentration being in the range of 100 and 300 nM.

3. Synthesis of standards and negative controls

4. Specificity analysis

2) The PCR amplification system of this example is shown in Table 5:

TABLE 5 10. mu.l PCR amplification System for the specific analysis of this example

Composition (I)	Volume of
		Magnetic bead PCR primer mixture	2.8μl
2 × Taq enzyme mix	5μl
		Template to be tested	2μl
ddH₂O	To 10. mu.l

3) fluorescence labeling: preparing SAPE mixed liquor, including 1.875 μ l of SAPE stock solution (Life Technologies, S-866); mu.l of 1 XTM hybridization solution (0.2M NaCl, 0.1M Tris, 0.08% Triton X-100, pH 8.0). The PCR product of step 2) was removed from the PCR machine and immediately placed on a magnetic plate (V & P Scientific, VP771LD-4CS) for 60s before the supernatant was discarded. And respectively adding 25 mu l of the prepared SAPE mixed solution into each reaction hole, sealing the membrane, and then shaking and uniformly mixing for 20 s. The PCR instrument was set up and the reaction was carried out as follows: 37 ℃ for 15 min. After the reaction, the 96-well plate was immediately placed on a magnetic plate for 60 seconds, and the supernatant was discarded. Adding 50 μ l of 1 XTM hybridization solution into each reaction well, shaking and mixing for 20s, placing on a magnetic plate for 30-60s, discarding the supernatant, and repeating for 1 time. Finally, 75. mu.l of 1 XTM hybridization solution was added to each reaction well, and the mixture was shaken and mixed for 20 seconds.

6) The detection results are shown in Table 6, wherein M-STEP9, M-VIM, M-FOXE1 and M-beta-ACTIN are target region standard products of methylation sites, and NC is negative standard product.

TABLE 6 detection results of specificity of magnetic bead-primer conjugates

Position of	Sample(s)	B39_STEP9
			1(1,A1)	M-VIM	25
2(1,B1)	M-FOXE1	37
			3(1,C1)	M-β-ACTIN	28
4(1,D1)	M-STEP9	719
			5(1,E1)	NC	42

Example 5 multiplex assay

1. The primers and probes of this example were synthesized by the biosciences of Kinsley; negative standards were bisulfite-converted human unmethylated DNA purchased from Zymo RESEARCH Bio (ZYMO RESEARCH, D5014); tissue extraction kit was purchased from blood/cell/tissue genomic DNA extraction kit (TIANGEN, DP304) produced by TIANGEN biochemical technology (beijing) ltd; 2 × Taq Master mix (required Taq enzyme, dNTP mix; MgCl2 and reaction buffer were prepared as a 2-fold concentration mix) the required reagents were purchased from TAKALA (TAKALA, RR 001B); SAPE stock solution was purchased from Life Technologies, S-866, seimer hewlett packard Technologies, china ltd, TMAC (tetramethylammonium chloride solution) was purchased from Sigma aldrich trade ltd (Sigma T3411); sarkosyl solution (sodium lauryl sarcosinate) was purchased from Sigma aldrich trade ltd (Sigma L7414); Tris-HCL Tris (hydroxymethyl) aminomethane hydrochloride, available from Solambio T8230, Inc., Beijing Sorley technologies, Inc.; triton X-100 (polyethylene glycol p-isooctylphenyl ether) was purchased from Solambio T8200, Kyoto Soilebao Tech., Ltd.; EDTA (ethylene diamine tetraacetic acid) was purchased from Seiko Biotech, Inc. (BioFroxx 1340GR 500); tris (Tris hydroxymethyl aminomethane) was purchased from Seitland Biotechnology GmbH (BioFroxx 1115GR 500); NaCl (sodium chloride) was purchased from Tianda chemical reagent.

An upstream primer:

FOXE1-M-F 5’Amino Modifier C12-TTTGTTCGTTTTTCGATTGTTC-3'(SEQ ID NO.3)

an upstream primer:

VIM-M-F 5’Amino Modifier C12-ATGTTACGCGTTTTTTTGTC-3'(SEQ ID NO.4)

an upstream primer:

β-ACTIN-M-F 5’Amino Modifier C12-TTTAATGTTACGTACGATTTTTC-3'(SEQ ID NO.5)

A downstream primer:

FOXE1-M-R 5’Biotin-TAACGCTATAAAACTCCTACCGC-3'(SEQ ID NO.6)

a downstream primer:

VIM-M-R 5’Biotin-AACTCCACCTTCTCGTTAATAC-3'(SEQ ID NO.7)

internal reference gene primers:

a downstream primer:

β-ACTIN-M-R 5’Biotin-GTAGGATGGTATGGGGGA-3'(SEQ ID NO.8)

where Biotin represents the Biotin modification.

The dry primers were dissolved by adding TE pH8.0 separately.

6. Multiplex assays

1) The PCR amplification system of this example is shown in Table 7 below:

TABLE 7 10. mu.l PCR amplification System for multiplex detection in this example

Composition (I)	Volume of
		Magnetic bead PCR primer mixture	2.8μl
2 × Taq enzyme mix	5μl
		DNA template after transformation	2μl
ddH₂O	To 10. mu.l

2) fluorescence labeling: preparing an SAPE mixed solution, including 5.25 μ l of an SAPE stock solution (Life Technologies, S-866); mu.l of 1 XTM hybridization solution (0.2M NaCl, 0.1M Tris, 0.08% Triton X-100, pH 8.0). The PCR product of step 1) was removed from the PCR machine and immediately placed on a magnetic plate (V & P Scientific, VP771LD-4CS) for 60s, and the supernatant was discarded. And respectively adding 25 mu l of the prepared SAPE mixed solution into each reaction hole, sealing the membrane, and then shaking and uniformly mixing for 20 s. The PCR instrument was set up and the reaction was carried out as follows: 37 ℃ for 15 min. After the reaction, the 96-well plate was immediately placed on a magnetic plate for 60 seconds, and the supernatant was discarded. Adding 50 μ l of 1 XTM hybridization solution into each reaction well, shaking and mixing for 20s, placing on a magnetic plate for 30-60s, discarding the supernatant, and repeating for 1 time. Finally, 75. mu.l of 1 XTM hybridization solution was added to each reaction well, and the mixture was shaken and mixed for 20 seconds.

7. The results are shown in Table 8, with NC as the negative control and PC as the positive control.

TABLE 8 multiple assay results for colorectal cancer samples in this example

Position of	Sample(s)	B39_STEP9	B55_FOXE1	B66_VIM	B72_β-ACTIN
						1(1，A1)	C1	480	790	873.5	834
2(1，B1)	C2	708.5	803	751	578
						3(1，C1)	C3	937	744.5	881	695.5
4(1，D1)	C4	98	631.5	988	812
						5(1，E1)	C5	75.5	523	27	572
6(1，F1)	H1	51	43	43	550
						7(1，F1)	H2	36	33.5	21	605
8(1，G1)	H3	38	40.5	118.5	507
						9(1，A2)	H4	51	43	43	715
10(1，B2)	H5	44	33.5	113	505
						11(1，C2)	PC	729	982.5	760	696
12(1，D2)	NC	35	38	46.5	60
						13(1，E2)	H	32	88.5	68	45

The detection results of several positive control sites are all more than 3 times of the NC value of the corresponding value, which proves that the method can simultaneously detect a plurality of sites; at least 2 sites of all 5 patient samples are positive, and all the controls are negative, so that the colorectal cancer samples can be well distinguished by the method.

The foregoing detailed description is provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications of the presently recited embodiments will be apparent to those of ordinary skill in the art and are intended to be within the scope of the appended claims and their equivalents.

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Claims

1. A method of methylation detection, comprising the steps of:

2. The method of claim 1, wherein the amino modification comprises having an amino modification functional group at the 5' end of the upstream primer, wherein the amino modification functional group comprises at least one free amino group.

3. The method of any one of claims 1-2, wherein the surface of the support has a hydroxyl modification.

4. The method according to any one of claims 2-3, wherein in the carrier-forward primer conjugate, the carrier and the forward primer are linked through the interaction of the amino group of the amino-modified functional group and the hydroxyl group on the surface of the carrier.

5. The method of any one of claims 1-4, wherein in the carrier-forward primer conjugate, the carrier is attached to the 5' end of the forward primer.

6. The method of any one of claims 1-5, wherein the label is capable of directly or indirectly visualizing an amplification product of the PCR amplification.

7. The method of any one of claims 1-6, wherein the label comprises a biotin label and/or a fluorescein label.

8. The method of any one of claims 1-7, wherein the sample is derived from a blood sample, a plasma sample, an FFPE sample, a tissue sample, a stool sample, and/or a urine sample.

9. A PCR primer pair capable of specifically amplifying a methylation site of the gene of interest, comprising an upstream primer and a downstream primer according to any one of claims 1-8.

10. A methylation detection kit comprising the forward primer of any one of claims 1-8, and/or the PCR primer pair of claim 9.