CN116790748A

CN116790748A - Method for detecting methylation sites

Info

Publication number: CN116790748A
Application number: CN202210267014.4A
Authority: CN
Inventors: 高蕾; 贺綦; 陈晨; 张若寒
Original assignee: Ankai Life Technology Suzhou Co ltd
Current assignee: Ankai Life Technology Suzhou Co ltd
Priority date: 2022-03-18
Filing date: 2022-03-18
Publication date: 2023-09-22

Abstract

The application relates to a methylation detection method, which comprises the following steps: a) Obtaining a probe and a PCR primer specific to a methylation site of a target gene, wherein the PCR primer comprises an upstream primer and a downstream primer, and the 5' end of the downstream primer is provided with a label; wherein the 5 'end of the probe is modified by amino, and the 5' end of the probe is coupled with a carrier; b) Carrying out PCR amplification on the sample by the upstream primer and the downstream primer to obtain a PCR amplification product; hybridizing the PCR amplification product with the probe to obtain a hybridization product; c) Detecting the signal of the hybridization product. The methylation detection method only needs one PCR amplification to detect the methylation sites of more than one gene with high specificity and high sensitivity, and has wide application value.

Description

Method for detecting methylation sites

Technical Field

The application relates to the field of biological medicine, in particular to a method for detecting a methylation site.

Background

Currently, DNA methylation is the most well-studied epigenetic mechanism, which is closely related to human tumorigenesis. For example, in the genome of a healthy person, cpG sites in CpG islands are usually in an unmethylated state. When the tumor occurs, the unmethylation degree of the CpG sequences outside the CpG island of the cancer suppressor gene is increased, and the CpG in the CpG island is in a highly methylated state.

Currently, there are several methods for studying DNA methylation levels: methylation sensitive restriction enzyme (MS-RE) method, methylation specific PCR (MS-PCR) method, methylation sensitive high resolution melting curve analysis (MS-HRM) method, binding Bisulfite Sequencing PCR (BSP) method or matrix assisted laser desorption ionization time of flight mass spectrometry (Massarray) method. However, these methods have problems of either very high requirements for the operation steps, primer design, instrument precision, etc. required for detection, or failure to learn methylation information of each CpG site, and also have problems of expensive, cumbersome and/or unfavorable for clinical large-scale rapid detection.

Therefore, a need exists for a method that is simple to operate and high in repeatability; can be applied to various sample types and multi-site DNA methylation detection methods in various fields.

Disclosure of Invention

The application provides a methylation detection method, and a primer pair and a kit for detecting methylation of a target gene. The method according to the application has at least the advantage of being selected from the group consisting of: 1. the method is simple and quick, does not need to cover the cover, and reduces the pollution risk of PCR products. 2. The specificity is strong, the sensitivity is high, and a plurality of indexes can be detected at one time. 3. Multiplex assays (e.g., simultaneous detection of multiple samples and/or multiple methylated gene loci) result in clear interpretation. 4. Suitable for use, but not limited to, human blood samples, plasma samples, FFPE samples, tissue samples, fecal samples, urine samples, and plant microbial samples. 5. Can be applied to multi-site DNA methylation detection in various fields, including but not limited to cancer screening, disease diagnosis and scientific research.

In one aspect, the present application provides a method of methylation detection comprising the steps of: a) Obtaining a probe and a PCR primer specific to a methylation site of a target gene, wherein the PCR primer comprises an upstream primer and a downstream primer, and the 5' end of the downstream primer is provided with a label; wherein the 5 'end of the probe is modified by amino, and the 5' end of the probe is coupled with a carrier; b) Carrying out PCR amplification on the sample by the upstream primer and the downstream primer to obtain a PCR amplification product; hybridizing the PCR amplification product with the probe to obtain a hybridization product; c) Detecting the signal of the hybridization product.

In certain embodiments, the gene of interest comprises a tumor specific gene.

In certain embodiments, the tumor comprises colorectal, gastric, and/or pancreatic cancer.

In certain embodiments, the gene of interest comprises SEPT9, FOXE1, VIM, RUNX3, P16, RASSFIA, APC, and/or NPTX2.

In certain embodiments, the gene of interest is selected from the group consisting of:

a) SEPT9, FOXE1, and VIM;

b) RUNX3, P16 and RASSFIA; and, a step of, in the first embodiment,

c) APC and NPTX2.

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 downstream primer comprises a nucleotide sequence set forth in any one of SEQ ID nos. 3,8, 10,15,19,21,26,27.

In certain embodiments, the concentration of the downstream primer is about 1. Mu.M to about 3. Mu.M.

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

In certain embodiments, the upstream primer comprises the nucleotide sequence set forth in any one of SEQ ID nos. 2,7,9,14,18,20,23,25.

In certain embodiments, the concentration of the upstream primer is about 1. Mu.M to about 3. Mu.M.

In certain embodiments, the upstream primer comprises the nucleotide sequence set forth in SEQ ID No. 2; and, the downstream primer comprises a nucleotide sequence shown in SEQ ID NO. 3; alternatively, the upstream primer comprises the nucleotide sequence shown as SEQ ID NO. 7; and, the downstream primer comprises a nucleotide sequence shown in SEQ ID NO. 8; alternatively, the upstream primer comprises the nucleotide sequence shown as SEQ ID NO. 9; and, the downstream primer comprises a nucleotide sequence shown as SEQ ID NO. 10; alternatively, the upstream primer comprises the nucleotide sequence shown as SEQ ID NO. 14; and, the downstream primer comprises a nucleotide sequence shown as SEQ ID NO. 15; alternatively, the upstream primer comprises the nucleotide sequence shown as SEQ ID NO. 18; and, the downstream primer comprises a nucleotide sequence shown as SEQ ID NO. 19; alternatively, the upstream primer comprises the nucleotide sequence shown as SEQ ID NO. 20; and, the downstream primer comprises a nucleotide sequence shown as SEQ ID NO. 21; alternatively, the upstream primer comprises the nucleotide sequence shown as SEQ ID NO. 23; and, the downstream primer comprises a nucleotide sequence shown as SEQ ID NO. 23; alternatively, the upstream primer comprises the nucleotide sequence shown as SEQ ID NO. 25; and, the downstream primer comprises the nucleotide sequence shown in SEQ ID NO. 26.

In certain embodiments, the amino modification comprises providing the 5' end of the probe with an amino-modifying functional group, wherein the amino-modifying functional group comprises at least one free amino group.

In certain embodiments, the amino-modifying functional group comprises a 5' amino C6 to C18.

In certain embodiments, the probe is at a concentration of about 1 μm to about 3 μm.

In certain embodiments, the probe comprises a nucleotide sequence set forth in any one of SEQ ID nos. 1,4,6,13,16,17,24.

In certain embodiments, the carrier is linked to the probe through the amino-modifying functional group.

In certain embodiments, the carrier is linked to the probe through an amino group in the amino-modifying functional group.

In certain embodiments, the carrier comprises magnetic beads and/or microspheres.

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

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

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

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

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

In certain embodiments, the PCR amplification of step b) comprises the following reaction steps: pre-denaturation at 95 ℃ for 2-5min, thermal circulation at 95 ℃ for 15-20s, thermal circulation at 52-60 ℃ for 20-30s, thermal circulation at 72 ℃ for 30s, thermal circulation at 30-45 s, and final extension at 72 ℃ for 5-10min;

in certain embodiments, the method further comprises the steps of: a) Obtaining an internal reference probe and an internal reference primer and an internal reference probe which are not specific to the methylation site of a target gene, wherein the internal reference primer comprises an internal reference upstream primer and an internal reference downstream primer, and the 5' end of the internal reference downstream primer is provided with a mark; wherein the 5 'end of the internal reference probe is modified by amino, and the 5' end of the internal reference probe is coupled with a carrier; b) Performing PCR amplification on the internal reference upstream primer and the internal reference downstream primer to obtain a PCR amplification product; hybridizing the PCR amplification product with the internal reference probe to obtain a hybridization product; c) Detecting the signal of the hybridization product.

In certain embodiments, the internal reference primer amplifies β -ACTIN.

In certain embodiments, the internal reference probe comprises the nucleotide sequence set forth in SEQ ID NO. 5.

In certain embodiments, the internal reference upstream primer comprises the nucleotide sequence set forth in SEQ ID No. 11; and/or, the internal reference downstream primer comprises a nucleotide sequence shown as SEQ ID NO. 12.

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 cells, tissues, organs and/or samples are derived from microorganisms, plants, animals and/or humans.

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

In certain embodiments, the sample of cells, tissue, organs and/or subjects is derived from a tumor patient and/or the cells, tissue comprise tumor cells and/or tumor tissue.

In certain embodiments, the method further comprises the step of: d) Obtaining methylation modification conditions of the sample according to the fluorescent signals.

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

In certain embodiments, the biotin is capable of binding to a streptavidin-labeled fluorescent dye to generate a fluorescent signal in the PCR amplification product.

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

In another aspect, the application also provides a primer pair comprising an upstream primer of the application and a downstream primer of the application.

In another aspect, the application also provides a kit comprising the upstream primer of the application and/or the downstream primer of the application.

In certain embodiments, the kit comprises a probe of the application.

Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the application as claimed. Accordingly, the drawings and descriptions of the present application are to be regarded as illustrative in nature and not as restrictive.

Drawings

The specific features of the application related to the application are shown in the appended claims. A better understanding of the features and advantages of the application in accordance with the present application will be obtained by reference to the exemplary embodiments and the accompanying drawings that are described in detail below. The brief description of the drawings is as follows:

FIG. 1 shows a schematic diagram of a coupling process of the capture magnetic beads according to the present application.

FIG. 2 shows a schematic flow chart of the methylation detection method of the present application.

Detailed Description

Further advantages and effects of the present application will become readily apparent to those skilled in the art from the present disclosure, by describing embodiments of the present application with specific examples.

Definition of terms

In the present application, the term "methylation" generally refers to a covalent bond between the carbon atom at position 5 of cytosine and a methyl group on DNA, whereby cytosine is modified to 5-methylcytosine (5 mC). The types of DNA methylation can be categorized as maintenance methylation (maintenance DNA methylation) and re-methylation (de novo methylation). Maintaining methylation refers to the process of methylation modification at the corresponding position of the daughter strand during the semi-preserved replication of the DNA by methyltransferase. Re-methylation refers to the process of methylation on a DNA duplex that was not previously methylated by a methyltransferase, after which a stable DNA methylation state is maintained by a maintenance methylase. The methylation may also include the formation of N6-methylpurines (N6-mA) and 7-methylguanines (7-mG). Abnormal changes in methylation are common in a variety of tumors, and abnormal DNA methylation status is one of the important features of tumors.

In the present application, the term "upstream primer" generally refers to a primer as a sense strand. The DNA molecule is double-stranded, wherein the 5'-3' strand is called the positive strand and the 3'-5' strand is called the negative strand. The strand synthesized by the upstream primer may be identical to the sequence of the forward strand. The strand synthesized by the upstream primer may be complementary to the sequence of the negative strand.

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

In the present application, the term "tumor-specific gene" generally refers to tumor-associated (TAA) as well as tumor-specific antigens (TSA). For example, the tumor-specific genes may include specific genes for solid tumors and/or non-solid tumors. A person skilled in the art can obtain a large number of potential such tumor-specific genes from databases, for example the TCGA database (https:// www.cancer.gov/about-nci/organization/ccg/research/construction-genetics/TCGA). The tumor-specific gene may include a molecular species that is an indicator of a disease or effect.

In the present application, the term "colorectal cancer" generally refers to medical conditions characterized by cancer cells of the intestinal tract below the small intestine (i.e., the large intestine (colon), including cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum), and may also include pre-cancerous lesions (also known as high-risk adenomas), early and late stage cancers. Furthermore, the term "colorectal cancer" may also include medical conditions characterized by cancer cells of the duodenum and small intestine (jejunum, ileum)

In the present application, the term "gastric cancer" generally refers to cancer of the stomach or stomach cells. These cancers may be adenocarcinomas that occur in the inner layer (mucosa) of the stomach and may occur in the pylorus, gastric body, or cardiac (inferior, body, and superior) portions of the stomach.

In the present application, the term "pancreatic cancer" generally refers to cancer derived from pancreatic cells. For example, the pancreatic cancer may be pancreatic adenocarcinoma. The concomitant symptoms of pancreatic cancer may be well known in medical standard textbooks such as Stedmen or pscryrembl.

In the present application, the term "SEPT9" generally refers to the septin9 gene, which may include the human SEPT9 gene. The accession number of the human SEPTIN9 gene in GenBank is 10801.

In the present application, the term "FOXE1" generally refers to the forkhead box E1 gene, which may include the human FOXE1 gene. The human FOXE1 gene has accession number 2304 in GenBank.

In the present application, the term "VIM" generally refers to a vimentin gene, which may include a human VIM gene. The accession number of human VIM gene in GenBank is 7431.

In the present application, the term "RUNX3" generally refers to the RUNX family transcription factor gene, which may include the human RUNX3 gene. The human RUNX3 gene has accession number 864 in GenBank.

In the present application, the term "P16" refers generally to the cyclin dependent kinase inhibitor a gene, which may include the human P16 gene. The accession number of the human P16 gene in GenBank is 1029.

In the present application, the term "RASSFIA" refers generally to the Ras association domain family member gene, which may include the human RASSFIA gene. The human RASSFIA gene has accession number 11186 in GenBank.

In the present application, the term "APC" generally refers to APC regulator of WNT signaling pathway gene, which may include human APC gene. The human APC gene has accession number 324 in GenBank.

In the present application, the term "NPTX2" refers generally to the neuronal pentraxin gene, which may include the human NPTX2 gene. The accession number of the human NPTX2 gene in GenBank is 4885.

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 have at least 80% (e.g., at least 85%, at least 90%, at least 95% or more) homology to the sequence of the 3'-5' strand of the DNA sequence near the methylation site of the gene of interest (i.e., the nucleotides at the upstream 5 'and downstream 3' of the methylation site).

In the present application, the term "upstream specific sequence" generally refers to a nucleotide sequence in the upstream primer that can specifically amplify a methylation site of a gene of interest. For example, the upstream specific sequence may have at least 80% (e.g., at least 85%, at least 90%, at least 95% or more) homology to the sequence of the 5'-3' strand of the DNA sequence near the methylation site of the gene of interest (i.e., the nucleotides at the upstream 5 'and downstream 3' of the methylation site).

In the present application, the term "probe" generally refers to a synthetic or biologically produced nucleic acid (DNA or RNA) containing a specific nucleotide sequence that allows it to specifically hybridize to a target nucleic acid sequence under defined stringency by design or selection. The oligonucleotide sequences used as detection probes may be labeled with a detectable group. Various labelling groups may be known in the art, for example radioactive, fluorescent, chemiluminescent or electrochemiluminescent compounds. In the present application, the probe may be modified, for example, at the 5' end with an amino group, so that it may be coupled to a carrier (for example, a magnetic bead).

In the present application, the term "hydroxy modification" generally refers to a modification that results in the modification of a functional group comprising a hydroxy group (e.g., free hydroxy group). For example, the hydroxyl modification includes modification such that it produces a free hydroxyl group at least at one end.

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

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

In the present application, the term "fluorescent label" generally refers to an intrinsically fluorescent compound, chemical group or composition. The fluorescent label may include a fluorophore that may contain a substituent that alters the solubility, spectral characteristics, or physical characteristics of the fluorophore. The person skilled in the art can select suitable fluorescent labels by labeling purposes, which may for example comprise FAM (carboxyfluorescein), CY3, HEX (hexachloro-6-methylfluorescein) and/or TAMRA (6-carboxytetramethyl rhodamine).

In the present application, the term "PCR" refers generally to a method whereby the concentration of a fragment of a target sequence in a mixture of genomic DNA can be increased without cloning or purification. The process of amplifying a target sequence may involve introducing two large excesses of oligonucleotide primers into a DNA mixture comprising the desired target sequence, followed by a precisely sequential thermal cycling in the presence of a DNA polymerase. The two primers may be complementary to the corresponding strands in the double stranded target sequence. To effect amplification, the mixture is denatured and the primers are then annealed to their complementary sequences within the target molecule. After annealing, the primer may be extended with a polymerase to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated multiple times (i.e., denaturation, annealing, and extension constitute one "cycle", and there can be many "cycles") to obtain high concentrations of amplified fragments of the desired target sequence. The length of the amplified fragments of the desired target sequence may be determined by the position of the primers relative to each other, and thus this length is a controllable parameter. By virtue of the reproducibility of this procedure, this method can be referred to as "polymerase chain reaction" (abbreviated as PCR).

In the present application, the term "liquid phase chip system" generally refers to a multifunctional suspended dot matrix instrument. The liquid phase chip system can be Multi-Analyte Suspension Array, MASA, flexible Multi-Analyte Profiling or xMAP. The liquid chip system can detect biological macromolecules such as proteins, nucleic acids 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-phase chip system can detect only a small amount of samples, and can simultaneously perform qualitative and quantitative detection.

In the present application, the term "primer pair" generally refers to a pair of nucleotide sequences that are capable of efficiently amplifying a template DNA. For example, the primer pair may include the upstream primer and the downstream primer.

Detailed Description

According to the methylation detection method, the methylation sites of the target genes are subjected to specific amplification and hybridization of amplification products and carriers are realized through one round of PCR, so that complicated steps of methylation detection are simplified, and the number of times of uncovering a sample in the detection process is reduced. In addition, the methylation detection method disclosed by the application is suitable for simultaneously detecting methylation sites of multiple source samples (such as from different individuals) and/or multiple target genes, and can greatly improve the detection efficiency. The methylation detection method is reliable in detection effect and good in repeatability.

Specifically, in the present application, the downstream primer may be labeled at the 5' end (e.g., biotin-labeled or fluorescent-labeled). PCR 1 rounds of amplification can be performed using the upstream primer and the downstream primer pair samples (e.g., samples that have been bisulfite treated). The amplification product obtained may be labeled (e.g., biotin-labeled or fluorescent-labeled). In the present application, the 5' end of the probe comprises amino modification and is coupled with a carrier (e.g., magnetic beads and/or microspheres), thereby obtaining a carrier-probe conjugate. The carrier-probe conjugate may hybridize to the amplification product (e.g., by hybridization of complementary sequences in the probe to each other) to obtain a hybridization product. The hybridization product may comprise both a coupled carrier (e.g., magnetic beads and/or microspheres) and a biotin label or a fluorescent label. For hybridization products containing both coupled carriers (e.g., magnetic beads and/or microspheres) and fluorescent labels, the fluorescent signals can be directly detected, and methylation detection results of corresponding samples can be obtained through the fluorescent signals; for hybridization products that include both coupled carriers (e.g., magnetic beads and/or microspheres) and biotin labels, a fluorescent signal can be generated using Streptavidin (SAPE) and methylation detection results can be obtained by the fluorescent signal.

In the present application, the method may perform (e.g., multiplex) methylation detection on more than one gene of interest simultaneously on a sample. In the present application, the methods may be used to predict, diagnose and/or assess the methylation level of one or more genes of interest in a sample. For example, it may be used to predict, diagnose, and/or assess the health of a sample (e.g., whether a tumor is present, the type of tumor is present, and/or the stage of the tumor is present).

In the present application, the target gene may include a tumor-specific gene. The tumor may comprise a solid tumor and/or a non-solid tumor.

In the present application, the tumor may include colorectal cancer, gastric cancer and/or pancreatic cancer. In the present application, the gene of interest may include specific genes associated with colorectal, gastric and/or pancreatic cancer, for example, specific genes known in the art that may aid in predicting, diagnosing and/or assessing the condition of colorectal, gastric and/or pancreatic cancer.

In the present application, the target gene may include SEPT9, FOXE1, VIM, RUNX3, P16, RASSFIA, APC and/or NPTX2. For example, the gene of interest may be selected from the group consisting of: a) SEPT9, FOXE1, and VIM; b) RUNX3, P16 and RASSFIA; and, c) APC and NPTX2.

In the present application, the 5' end of the downstream primer may be labeled. For example, the label can directly or indirectly color the PCR amplification product.

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

In the present application, the downstream primer may comprise a downstream specific sequence capable of specifically amplifying the methylation site of the target gene. For example, the downstream primer may comprise a downstream specific sequence capable of specifically amplifying the methylation sites of the SEPT9, FOXE1, VIM, RUNX3, P16, RASSFIA, APC and/or NPTX2 genes.

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

For example, the downstream primer may comprise the nucleotide sequence set forth in any one of SEQ ID nos. 3,8,10,15,19,21,26,27.

In the present application, the concentration of the downstream primer may be about 1. Mu.M to about 3. Mu.M. For example, the concentration may be from about 1.2. Mu.M to about 3. Mu.M, from about 1.4. Mu.M to about 3. Mu.M, from about 1.6. Mu.M to about 3. Mu.M, from about 1.8. Mu.M to about 3. Mu.M, from about 2.0. Mu.M to about 3. Mu.M, from about 1. Mu.M to about 2.8. Mu.M, from about 1. Mu.M to about 2.5. Mu.M, from about 1.5. Mu.M to about 3. Mu.M, from about 1.5. Mu.M to about 2.5. Mu.M, or from about 1.5. Mu.M to about 2. Mu.M.

In the present application, the upstream primer may comprise an upstream specific sequence capable of specifically amplifying the methylation site of the target gene. For example, the upstream primer may comprise an upstream specific sequence capable of specifically amplifying the methylation sites of the SEPT9, FOXE1, VIM, RUNX3, P16, RASSFIA, APC and/or NPTX2 genes.

In the present application, the upstream primer may comprise a nucleotide sequence shown in any one of SEQ ID NO.2,7,9,14,18,20,23,25.

In the present application, the concentration of the upstream primer may be about 1. Mu.M to about 3. Mu.M. For example, the concentration may be from about 1.2. Mu.M to about 3. Mu.M, from about 1.4. Mu.M to about 3. Mu.M, from about 1.6. Mu.M to about 3. Mu.M, from about 1.8. Mu.M to about 3. Mu.M, from about 2.0. Mu.M to about 3. Mu.M, from about 1. Mu.M to about 2.8. Mu.M, from about 1. Mu.M to about 2.5. Mu.M, from about 1.5. Mu.M to about 3. Mu.M, from about 1.5. Mu.M to about 2.5. Mu.M, or from about 1.5. Mu.M to about 2. Mu.M.

In the present application, the upstream primer may be matched with the downstream primer to amplify the target gene. In the present application, the upstream primer may comprise the nucleotide sequence shown in SEQ ID NO. 2; and, the downstream primer may comprise the nucleotide sequence shown in SEQ ID NO. 3; alternatively, the upstream primer may comprise the nucleotide sequence shown as SEQ ID NO. 7; and, the downstream primer may comprise the nucleotide sequence shown in SEQ ID NO. 8; alternatively, the upstream primer may comprise the nucleotide sequence shown as SEQ ID NO. 9; and, the downstream primer may comprise the nucleotide sequence shown in SEQ ID NO. 10; alternatively, the upstream primer may comprise the nucleotide sequence shown as SEQ ID NO. 14; and, the downstream primer may comprise the nucleotide sequence shown in SEQ ID NO. 15; alternatively, the upstream primer may comprise the nucleotide sequence shown as SEQ ID NO. 18; and, the downstream primer may comprise the nucleotide sequence shown in SEQ ID NO. 19; alternatively, the upstream primer may comprise the nucleotide sequence shown as SEQ ID NO. 20; and, the downstream primer may comprise the nucleotide sequence shown in SEQ ID NO. 21; alternatively, the upstream primer may comprise the nucleotide sequence shown as SEQ ID NO. 23; and, the downstream primer may comprise the nucleotide sequence shown in SEQ ID NO. 23; alternatively, the upstream primer may comprise the nucleotide sequence shown as SEQ ID NO. 25; and, the downstream primer may comprise the nucleotide sequence shown as SEQ ID NO. 26.

In the present application, the 5 'end of the probe has an amino modification, and the 5' end of the probe is coupled to a carrier. In the present application, the amino modification may include providing the 5' end of the probe with an amino-modifying functional group, wherein the amino-modifying functional group may include 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 an Amino Modifier C12. The Amino Modifier C12 may have the following structure:

in the present application, the concentration of the probe may be about 1. Mu.M to about 3. Mu.M. For example, the concentration may be from about 1.2. Mu.M to about 3. Mu.M, from about 1.4. Mu.M to about 3. Mu.M, from about 1.6. Mu.M to about 3. Mu.M, from about 1.8. Mu.M to about 3. Mu.M, from about 2.0. Mu.M to about 3. Mu.M, from about 1. Mu.M to about 2.8. Mu.M, from about 1. Mu.M to about 2.5. Mu.M, from about 1.5. Mu.M to about 3. Mu.M, from about 1.5. Mu.M to about 2.5. Mu.M, or from about 1.5. Mu.M to about 2. Mu.M.

In the present application, the probe may hybridize with the PCR amplification product to obtain a hybridization product. For example, the probe may have at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, or more) complementarity to at least a portion of the sequences in the PCR amplification product to form a hybridization product. For example, the probe may have at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, or more) complementarity to at least a portion of the sequence associated with the methylation site of the gene of interest to form a hybridization product. In the present application, the probe may comprise a nucleotide sequence shown in any one of SEQ ID NO.1,4,6,13,16,17,24.

In the present application, the carrier and the probe may be linked through the amino-modifying functional group. For example, the carrier and the probe may be linked by an amino group in the amino-modifying functional group.

In the present application, the carrier may include a solid phase carrier and/or a liquid phase carrier. 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 carboxyl or amino modifications. The magnetic beads and/or microspheres may be selected from the products of Miltenyi, dynabeads, usa, magservice 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 hydroxyl group modification. For example, the carrier may be linked to the amino group of the amino-modifying functional group of the probe through a hydroxyl group in the hydroxyl-modifying functional group.

In the present application, the number of PCR amplifications as described in step b) may be 1.

In the present application, the PCR amplification of step b) may comprise the following reaction steps: pre-denaturation at 95 ℃ for 2-5min, thermal circulation at 95 ℃ for 15-20s, thermal circulation at 52-60 ℃ for 20-30s, thermal circulation at 72 ℃ for 5-30s, thermal circulation at 30-45 min, and final extension at 72 ℃ for 5-10min. For example, the PCR amplification may comprise the following reaction steps: pre-denaturation at 95℃for 2min, thermal cycling at 95℃for 15s,58℃for 22s,72℃for 30s,40 cycles, and final extension at 72℃for 5min. For example, the PCR amplification may comprise the following reaction steps: pre-denaturation at 95℃for 2min, thermal cycling at 95℃for 15s,60℃for 22s,72℃for 30s,35 cycles, and final extension at 72℃for 5min. For example, the PCR amplification may comprise the following reaction steps: pre-denaturation at 95℃for 2min, thermal cycling at 95℃for 15s,59℃for 22s,72℃for 30s,37 cycles, and final extension at 72℃for 5min.

In the present application, the method may further include the steps of: a) Obtaining an internal reference probe and an internal reference primer and an internal reference probe which are not specific to the methylation site of a target gene, wherein the internal reference primer can comprise an internal reference upstream primer and an internal reference downstream primer, and the 5' end of the internal reference downstream primer is provided with a label; wherein the 5 'end of the internal reference probe is modified by amino, and the 5' end of the internal reference probe is coupled with a carrier; b) Performing PCR amplification on the internal reference upstream primer and the internal reference downstream primer to obtain a PCR amplification product; hybridizing the PCR amplification product with the internal reference probe to obtain a hybridization product; c) Detecting the signal of the hybridization product.

In the present application, the internal reference primer may amplify β -ACTIN. For example, the reference probe may comprise the nucleotide sequence shown in SEQ ID NO. 5. For example, the reference upstream primer may comprise the nucleotide sequence shown in SEQ ID NO. 11; and/or the internal reference downstream primer may comprise the nucleotide sequence shown as SEQ ID NO. 12.

In the present application, the DNA in the sample may be bisulfite treated and/or enzymatically converted. In the present application, the DNA in the sample may be subjected to bisulfite treatment or enzymatic conversion. The method can convert all unmethylated cytosines to uracil, which can be converted to thymine in subsequent PCR amplifications. While the methylated cytosine remains unchanged during this process. The bisulfite treatment or enzymatic conversion can 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 include cells, tissues, organs and/or samples.

In the present application, the cells, tissues, organs and/or samples may be derived from microorganisms, plants, animals and/or humans.

In the present application, the sample may be derived from a blood sample, a plasma sample, an FFPE sample, a tissue sample, a fecal sample, and/or a urine sample.

In the present application, the cells, tissues, organs and/or samples of the subject may be derived from a tumor patient, and/or the cells, tissues may include tumor cells and/or tumor tissue. For example, the cells, tissue, organ, and/or sample of the subject may be derived from a tumor patient, and/or the cells, tissue include tumor cells and/or tumor tissue. For example, the tumor may include a solid tumor and/or a non-solid tumor. The methods of the application may be adapted to different indication patients and/or to different sources of samples obtained from the patients.

In the present application, the method may further include the steps of: d) Obtaining methylation modification conditions of the sample according to the fluorescent signals.

In the present application, step c) may comprise detecting and performing data analysis on fluorescent signals generated by said hybridization products comprising said carrier and fluorescent markers (e.g. hybridization products with magnetic beads and/or microsphere-fluorescent markers).

In the present application, step c) may comprise subjecting the hybridization products comprising the carrier and fluorescent label (e.g. hybridization products with magnetic beads and/or microsphere-biotin labels) to detection and data analysis. For example, the hybridization products with magnetic beads and/or microsphere-biotin labels may be fluorescently labeled using streptavidin-labeled fluorescent dyes (SAPE). In the present application, the reaction conditions of the fluorescent label may be about 10 to about 30 minutes at about 25 to about 37 ℃.

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

In the present application, in the PCR amplification product, the biotin is capable of binding to a streptavidin-labeled fluorescent dye to generate a fluorescent signal.

In the present application, the fluorescent label is capable of generating a fluorescent signal in the PCR amplification product.

In the present application, a biotin-streptavidin (or avidin) system may be used to cause the PCR amplification product to generate a fluorescent signal. For example, the magnetic beads and/or microspheres may be coated with streptavidin, which magnetic beads and/or microspheres may bind to the PCR amplification product to generate a fluorescent signal. In some cases, other different affinity binding partners may be used instead of the biotin-streptavidin (or avidin) system, e.g., antigens/haptens and antibodies, or enzymes and corresponding substrates may be used.

In the present application, the kit may comprise the probe of the present application. The kit can be used for realizing methylation detection of more than one target gene through one round of PCR. The kit of the application can be used for predicting, diagnosing and/or assessing the methylation level of more than one target gene of a sample. For example, it may be used to predict, diagnose, and/or assess the health of a sample (e.g., whether a tumor is present, the type of tumor is present, and/or the stage of the tumor is present). For example, it may be useful to predict, diagnose and/or assess the condition of a sample having colorectal, gastric and/or pancreatic cancer.

In the present application, the probe, the upstream primer and/or the downstream primer may be packaged in different independent packages.

In the present application, the kit may further comprise any reagent required for PCR amplification. For example, taq Master mix may be included.

In the present application, the kit may further comprise any reagent required for sample collection and/or processing. For example, a solution comprising bisulfite may be included.

The application also relates to the following embodiments:

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

a) Obtaining a probe and a PCR primer specific to a methylation site of a target gene, wherein the PCR primer comprises an upstream primer and a downstream primer, and the 5' end of the downstream primer is provided with a label; wherein the 5 'end of the probe is modified by amino, and the 5' end of the probe is coupled with a carrier;

b) Carrying out PCR amplification on the sample by the upstream primer and the downstream primer to obtain a PCR amplification product; hybridizing the PCR amplification product with the probe to obtain a hybridization product;

c) Detecting the signal of the hybridization product.

2. The method of embodiment 1, wherein the gene of interest comprises a tumor-specific gene.

3. The method of embodiment 2, wherein the tumor comprises colorectal, gastric, and/or pancreatic cancer.

4. The method of any one of embodiments 1-3, wherein the gene of interest comprises SEPT9, FOXE1, VIM, RUNX3, P16, RASSFIA, APC, and/or NPTX2.

5. The method of any one of embodiments 1-4, wherein the gene of interest is selected from the group consisting of:

a) SEPT9, FOXE1, and VIM;

b) RUNX3, P16 and RASSFIA; and, a step of, in the first embodiment,

c) APC and NPTX2.

6. The method of any one of embodiments 1-5, wherein the downstream primer comprises a downstream specific sequence capable of specifically amplifying the methylation site of the gene of interest.

7. The method of any one of embodiments 1-6, wherein the downstream primer comprises a nucleotide sequence set forth in any one of SEQ ID nos. 3,8, 10,15,19,21,26,27.

8. The method of any of embodiments 1-7, wherein the concentration of the downstream primer is about 1 μΜ to about 3 μΜ.

9. The method of any one of embodiments 1-8, wherein the upstream primer comprises an upstream specific sequence capable of specifically amplifying a methylation site of the gene of interest.

10. The method of any one of embodiments 1-9, wherein the upstream primer comprises a nucleotide sequence set forth in any one of SEQ ID No.2,7,9,14,18,20,23,25.

11. The method of any of embodiments 1-10, wherein the concentration of the upstream primer is about 1 μΜ to about 3 μΜ.

12. The method of any one of embodiments 1-11, wherein:

the upstream primer comprises a nucleotide sequence shown as SEQ ID NO. 2; and, the downstream primer comprises a nucleotide sequence shown in SEQ ID NO. 3; or alternatively, the process may be performed,

the upstream primer comprises a nucleotide sequence shown as SEQ ID NO. 7; and, the downstream primer comprises a nucleotide sequence shown in SEQ ID NO. 8; or alternatively, the process may be performed,

the upstream primer comprises a nucleotide sequence shown as SEQ ID NO. 9; and, the downstream primer comprises a nucleotide sequence shown as SEQ ID NO. 10; or alternatively, the process may be performed,

the upstream primer comprises a nucleotide sequence shown as SEQ ID NO. 14; and, the downstream primer comprises a nucleotide sequence shown as SEQ ID NO. 15; or alternatively, the process may be performed,

the upstream primer comprises a nucleotide sequence shown as SEQ ID NO. 18; and, the downstream primer comprises a nucleotide sequence shown as SEQ ID NO. 19; or alternatively, the process may be performed,

the upstream primer comprises a nucleotide sequence shown as SEQ ID NO. 20; and, the downstream primer comprises a nucleotide sequence shown as SEQ ID NO. 21; or alternatively, the process may be performed,

the upstream primer comprises a nucleotide sequence shown as SEQ ID NO. 23; and, the downstream primer comprises a nucleotide sequence shown as SEQ ID NO. 23; or alternatively, the process may be performed,

The upstream primer comprises a nucleotide sequence shown as SEQ ID NO. 25; and, the downstream primer comprises the nucleotide sequence shown in SEQ ID NO. 26.

13. The method of any one of embodiments 1-12, wherein the amino modification comprises providing the 5' end of the probe with an amino-modifying functional group, wherein the amino-modifying functional group comprises at least one free amino group.

14. The method of embodiment 13, wherein the amino-modifying functional group comprises 5' amino C6-C18.

15. The method of any one of embodiments 1-14, wherein the concentration of the probe is about 1 μm to about 3 μm.

16. The method of any one of embodiments 1-15, wherein the probe comprises a nucleotide sequence set forth in any one of SEQ ID nos. 1,4,6,13,16,17,24.

17. The method of any one of embodiments 1-16, wherein the carrier is linked to the probe through the amino-modifying functional group.

18. The method of any one of embodiments 1-17, wherein the carrier is linked to the probe through an amino group in the amino-modifying functional group.

19. The method of any one of embodiments 1-18, wherein the carrier comprises magnetic beads and/or microspheres.

20. The method of any one of embodiments 1-19, wherein the surface of the support has hydroxyl modifications.

21. The method of any one of embodiments 1-20, wherein the label is capable of directly or indirectly developing the PCR amplification product.

22. The method of any one of embodiments 1-21, wherein the tag is directly or indirectly linked to the 5' end of the downstream specific sequence.

23. The method of any one of embodiments 1-22, wherein the label comprises a biotin label and/or a fluorescent label.

24. The method of any one of embodiments 1-23, wherein the number of PCR amplifications of step b) is 1.

25. The method of any one of embodiments 1-24, wherein the PCR amplification of step b) comprises the following reaction steps: pre-denaturation at 95 ℃ for 2-5min, thermal circulation at 95 ℃ for 15-20s, thermal circulation at 52-60 ℃ for 20-30s, thermal circulation at 72 ℃ for 5-30s, thermal circulation at 30-45 min, and final extension at 72 ℃ for 5-10min;

26. the method of any one of embodiments 1-25, wherein the method further comprises the step of:

a) Obtaining an internal reference probe and an internal reference primer and an internal reference probe which are not specific to the methylation site of a target gene, wherein the internal reference primer comprises an internal reference upstream primer and an internal reference downstream primer, and the 5' end of the internal reference downstream primer is provided with a mark; wherein the 5 'end of the internal reference probe is modified by amino, and the 5' end of the internal reference probe is coupled with a carrier;

b) Performing PCR amplification on the internal reference upstream primer and the internal reference downstream primer to obtain a PCR amplification product; hybridizing the PCR amplification product with the internal reference probe to obtain a hybridization product;

c) Detecting the signal of the hybridization product.

27. The method of embodiment 26, wherein the internal reference primer amplifies β -ACTIN.

28. The method of any one of embodiments 26-27, wherein the internal reference probe comprises the nucleotide sequence set forth in SEQ ID No. 5.

29. The method of any one of embodiments 26-28, wherein the internal reference upstream primer comprises the nucleotide sequence set forth in SEQ ID No. 11; and/or, the internal reference downstream primer comprises a nucleotide sequence shown as SEQ ID NO. 12.

30. The method of any one of embodiments 1-29, wherein DNA in the sample is bisulfite treated and/or enzymatically converted.

31. The method of any one of embodiments 1-30, wherein the sample comprises genomic DNA, plasmid DNA, mitochondrial DNA, episomal DNA, and/or synthetic DNA.

32. The method of any one of embodiments 1-31, wherein the source of the sample comprises a cell, tissue, organ, and/or sample.

33. The method of embodiment 32, wherein the cells, tissues, organs and/or samples are derived from microorganisms, plants, animals and/or humans.

34. The method of any one of embodiments 1-32, 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.

35. The method of any one of embodiments 33-34, wherein the sample of cells, tissue, organs, and/or subjects is derived from a tumor patient, and/or the cells, tissue comprise tumor cells and/or tumor tissue.

36. The method of any one of embodiments 1-35, further comprising the step of:

d) Obtaining methylation modification conditions of the sample according to the fluorescent signals.

37. The method of embodiment 36, wherein the detecting comprises detecting using a liquid phase chip platform.

38. The method of any one of embodiments 23-37, wherein the biotin is capable of binding to a streptavidin-labeled fluorescent dye to generate a fluorescent signal in the PCR amplification product.

39. The method of any one of embodiments 23-37, wherein the fluorescent label is capable of generating a fluorescent signal in the PCR amplification product.

40. A primer pair comprising the upstream primer of any one of embodiments 1-39 and the downstream primer of any one of embodiments 1-39.

41. A kit comprising the upstream primer of any one of embodiments 1-39, and/or the downstream primer of any one of embodiments 1-39.

42. The kit of embodiment 41, comprising the probe of any one of embodiments 1-39.

Without intending to be limited by any theory, the following examples are merely illustrative of the methylation detection methods and uses of the present application, and are not intended to limit the scope of the present application.

Examples

Example 1 magnetic bead probe coupling

1. The probes used in this example were synthesized by the Kirschner Biotech company. Reagent MES (2- (N-morpholinoethanesulfonic acid) was purchased from Sigma Aldrich (Shanghai) trade Co., ltd. (Sigma M3671). EDAC N' - (ethyliminomethylene) -N, N-dimethyl-1, 3-propanediamine monohydrochloride was purchased from Sigma Aldrich (Shanghai) trade company, inc. (Sigma E7750). Tween-20 (Tween-20) was purchased from Beijing Soy Bao technology Co., ltd (Solarbio T8220). SDS (sodium dodecyl sulfate) was purchased from Beijing Soy Bao technology Co., ltd (Solarbio S8010). TE solution at pH 8.0: the composition of the material was 10mM Tris-HCl, 1mM EDTA.

2. A specific single-stranded DNA probe is designed aiming at a nucleic acid sequence of a region to be detected, and the 5' -end is modified by an Amino Modifier C12, so that the Amino group of the probe is coupled with the hydroxyl group of the microbead.

The length of the probe sequence is 30-45bp, and the TM value is 75-90 DEG C

The probe sequence is as follows:

TZ-VIM 5'Amino Modifier C12-TACGCGTTTTTTTGTCGTGCGTTTGCGGAGTAGCG-3'(SEQ ID NO.1)

the probe was diluted to 0.1nanomole with pure water for use.

3. The microbeads are MagPlax magnetic beads with surface hydroxyl modified by Luminex company, and are suitable for MAGPIX or Luminex100/200 liquid phase suspension chip systems by Luminex company. Other hydroxyl surface modified magnetic beads and other liquid phase chip systems may be used, and are not limited in this regard.

4. Coupling step

1) Uncoupled MagPlax magnetic beads 1.25X10 were taken ⁶ And adding the mixture into a 1.5ml centrifuge tube, magnetically sucking the mixture in a magnetic rack for 2min, and sucking the supernatant.

2) 10. Mu.L of 0.1M MES, pH 4.5 was added, mixed well with shaking and sonicated for 20s.

3) 2. Mu.L of probe (0.1 nanomole) was added to the tube and vortexed.

4) Fresh 10mg/ml EDAC was prepared, 0.75. Mu.L was added to the centrifuge tube, vortexed and left to stand in the dark for 30min.

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

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

7) 1ml of SDS solution with the volume percentage of 0.1% is added and placed on a magnetic rack for magnetic attraction for 2min, and the supernatant is scraped.

8) 50. Mu.L of TE solution (wherein the material composition is 10mM Tris-HCl, 1mM EDTA) at pH8.0 was added and vortexed to mix well to obtain a coupled magnetic bead-probe conjugate.

The number of microbeads is counted by using a hemocytometer, and if the microbeads are required to be stored, the microbeads should be stored at 2-8 ℃ in a dark place.

Example 2 assay specific assay

1. The primers used in this example were synthesized by Kirschner, inc., and plasmids containing the target region of the methylation site of M-VIM, M-SEPT9, M-FOXE1, β -ACTIN and NC (target region without methylation site) were synthesized by Jin Weizhi Biotechnology Co., ltd; 2 XTaq Master mix (Taq enzyme, dNTP mix, mgCl required) ₂ The reaction buffer was pre-formulated as a 2-fold concentration mixture) and the required reagents were purchased from TAKALA corporation (TAKALA, RR 001B); SAPE stock was purchased from sameiser femoris technologies (china) limited (Life Technologies, S-866); TMAC (tetramethyl ammonium chloride solution) was purchased from Sigma aldrich (Shanghai) trade company (Sigma T3411); sarkosyl solution (sodium dodecyl sarcosinate solution) was purchased from Sigma aldrich (Shanghai) trade company (Sigma L7414); tris-HCl Tris (hydroxymethyl) aminomethane hydrochloride was purchased from Beijing Soy technologies Inc. (Solarbio T8230); triton X-100 (polyethylene glycol p-isooctylphenyl ether) was purchased from Beijing Soy Bao technology Co., ltd (Solarbio T8200); EDTA (ethylenediamine tetraacetic acid) was purchased from Sairo BioFrox 1340GR 500; tris (Tris) is purchased from the Sairo BioFrox 1115GR 500); naCl (sodium chloride) was purchased from tianda chemical.

TE solution at pH 8.0: the composition of the material was 10mM Tris-HCl, 1mM EDTA.

2. Primer probe design and synthesis

The PCR primer is designed aiming at methylation sites, the purity of the primer reaches electrophoresis grade (PAGE) or HPLC grade, the length of the primer sequence is 18-35bp, the TM value is 50-60 ℃, and the 5' end of the downstream primer is subjected to biotin modification.

An upstream primer: VIM-M-F5'-ATGTTACGCGTTTTTTTGTC-3' (SEQ ID NO. 2)

A downstream primer: VIM-M-R5 '-Biotin-AACTCCACCTTCTCGTTAATAC-3' (SEQ ID NO. 3)

Wherein Biotin represents a Biotin modification.

The primer dry powder was dissolved by adding TE pH8.0 to make the concentration of the primer 2.5. Mu.M.

3. Synthesis of standard and negative reference

Dissolving dry powder of target region standard of M-VIM, M-SEPT9, M-FOXE1, M-beta-ACTIN methylation site and NC negative control standard in enzyme-free water, respectively, and diluting to 10 ¹⁵ Copy/ml, 10-fold gradient dilution of standard, 10 each ¹⁰ Copy/ml was used as a template for standard, and the specificity of the VIM bead-probe conjugate and VIM primer was detected using M-VIM, M-SEPT9, M-FOXE1, M- β -ACTIN, respectively.

4. Specificity analysis

1) M-VIM, M-SEPT9, M-FOXE1, M-beta-ACTIN methylation site target region standard, NC negative standard, and diluting according to the requirements to be used 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 in example 2 specificity analysis

Composition of the components	Volume of
		PCR primer (2.5. Mu.M)	0.8μl
2×Taq Master mix	5μl
		Template to be measured	2μl
ddH ₂ O	To 10 μl

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

3) Hybridization: mu.l of the PCR product of step 2) was taken, 33. Mu.l of 1.5 Xhybridization buffer (4.5M TMAC,0.15%Sarkosyl solution,75mM Tris-HCl,6mM EDTA,pH8.0), hybridization bead mix (Luminex,microheres) 4. Mu.l (2500 magnetic bead-probe conjugates per reaction), 11. Mu.l of water were mixed and shaken well. The PCR instrument was set up and reacted as follows: 96 ℃ 90s,58 ℃ 60min.

4) Color development: preparing a SAPE mixed solution comprising 1.875 μl of SAPE stock solution; 1×Tm hybridization solution (0.2M NaCl,0.1M Tris,0.08%Triton X-100, pH 8.0) 125. Mu.l. Immediately after removing the hybridization product from step 3) from the PCR apparatus, the supernatant was discarded after 60s on a magnetic plate (V & P Scientific, VP771LD-4 CS). And respectively adding 25 mu l of the prepared SAPE mixed solution into each reaction hole, sealing the film, and vibrating and uniformly mixing for 20s. The PCR instrument was set up and reacted as follows: 37℃for 15min. Immediately after the reaction, the 96-well plate was placed on a magnetic plate for 60s, and the supernatant was discarded. 50 μl of 1×Tm hybridization solution was added to each reaction well, followed by shaking and mixing for 20s, and then the supernatant was discarded after 30-60s in a magnetic plate, and repeated 1 time. Finally, 75 μl of 1×Tm hybridization solution was added to each reaction well, and mixed by shaking for 20s.

5) And (3) detecting a liquid-phase chip platform: and 4) detecting the product obtained in the step 4) by using a Luminex200 liquid-phase chip platform, wherein the detection operation steps and parameter setting are operated according to a Luminex200 operation instruction.

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

5. The detection results are shown in Table 2, M-VIM, M-SEPT9, M-FOXE1, M-beta-ACTIN are target region standard substances of methylation sites, and NC is a negative standard substance.

TABLE 2 magnetic bead-probe conjugate specificity detection results

Site(s)	Sample of	B66_VIM
			66(1,B9)	M-SEPT9	71
67(1,C9)	M-FOXE1	85.5
			68(1,D9)	M-β-ACTIN	74
69(1,E9)	M-VIM	2726
			90(1,F9)	NC	62

Therefore, the detection method VIM primer and the magnetic bead-probe conjugate can specifically distinguish M-VIM standard substances, which proves that the method has better specificity.

Example 3 multiplex detection assay

1. The primers and probes used in this example were synthesized by the gold srey biotechnology company; the positive and negative standards were human methylated DNA and human unmethylated DNA (zymesearch, D5014) after bisulfite conversion, purchased from Zymo RESEARCH biosystems (Zymo RESEARCH, D5014); the extraction of the fecal DNA sample is carried out by the self-matching reagent in the laboratory; 2 XTaq Master mix (Taq enzyme, dNTP mix required; mgCl) ₂ The reaction buffer was pre-formulated as a 2-fold concentration mixture) and the required reagents were purchased from TAKALA corporation (TAKALA, RR 001B); SAPE stock solution was purchased from Semer Feishier technologies (China) Co., ltd (Life Technologies, S-866), TMAC (tetramethyl ammonium chloride solution) was purchased from Sigma Aldrich trade Co., shanghaiCompany limited (Sigma T3411); sarkosyl solution (sodium dodecyl sarcosinate solution) was purchased from Sigma aldrich (Shanghai) trade company (Sigma L7414); tris-HCl Tris (hydroxymethyl) aminomethane, purchased from beijing solebao technologies limited (Solarbio T8230); triton X-100 (polyethylene glycol p-isooctylphenyl ether) was purchased from Beijing Soy Bao technology Co., ltd (Solarbio T8200); EDTA (ethylenediamine tetraacetic acid) was purchased from Sairo BioFrox 1340GR 500; tris (Tris) is purchased from the Sairo BioFrox 1115GR 500); naCl (sodium chloride) was purchased from tianda chemical.

2. The probe design and synthesis were performed according to the method described in example 1, and the sequences were as follows

TZ-SEPT9 5'Amino Modifier C12-TCGGTTTTTATATTCGTTTATATTTGGTCGTAGCGGGGCGTTCGG-3'(SEQ ID NO.4)；

TZ-β-actin 5'Amino Modifier C12-TCGTGGTGGTGAAGTTGTAGTCGCGTTCGGTGAGG-3'(SEQ ID NO.5)；

TZ-FOXE1 5'Amino Modifier C12-CGTCTCGTCGGGGTTCGGGCGTATTTTTTTAGGTAGGCGAGACG-3'(SEQ ID NO.6)。

The bead-probe coupling was performed as described in example 1, and the coupled bead-probe conjugate was subjected to a cell counter to count the number of beads, and a hybridization bead-probe mixture was prepared by using TE pH 8.0.

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

an upstream primer: SEPT 9-M-F5'-TTTTCGTTATGGTTCGGTTT-3' (SEQ ID NO. 7)

Downstream primer SEPT 9-M-R5 'Biotin-CTAAAAAACAATCCTAAACACAC-3' (SEQ ID NO. 8)

An upstream primer: FOXE 1-M-F5'-TTTGTTCGTTTTTCGATTGTTC-3' (SEQ ID NO. 9)

Downstream primer FOXE 1-M-R5 'Biotin-TAACGCTATAAAACTCCTACCGC-3' (SEQ ID NO. 10)

Internal reference gene primer:

an upstream primer: beta-ACTIN-M-F5'-TTTAATGTTACGTACGATTTTTC-3' (SEQ ID NO. 11)

A downstream primer: beta-ACTIN-M-R5 'Biotin-GTAGGATGGTATGGGGGA-3' (SEQ ID NO. 12).

Wherein Biotin represents a Biotin modification.

The primer dry powder is respectively added into TE pH8.0 for dissolution to prepare PCR primer mixed solution, so that the concentration of each pair of primers is 2.5 mu M.

4. Sample extraction: stool sample DNA was extracted from the self-assembling reagent and DNA extraction was performed on 5 human colorectal cancer stool samples and 5 healthy human stool samples.

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

6. Multiplex detection

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

TABLE 3 10. Mu.l PCR amplification System in multiplex detection example 3

Composition of the components	Volume of
		PCR primer mix (2.5. Mu.M)	0.8μl
2 XTaq enzyme mix	5μl
		Post-transformation DNA templates	2μl
ddH ₂ O	To 10 μl

2) Hybridization: mu.l of the PCR product of step 1) was taken, 33. Mu.l of 1.5 Xhybridization buffer (4.5M TMAC,0.15%Sarkosyl solution,75mM Tris-HCl,6mM EDTA,pH8.0), hybridization bead-probe mixture (Luminex,microsphere) 4. Mu.l (2500 per each bead-probe conjugate) and 11. Mu.l water were mixed and shaken well. The PCR instrument was set up and reacted as follows: 96 ℃ 90s,58 ℃ 60min.

3) Color development: preparing a SAPE mixed solution, wherein the SAPE mixed solution comprises 5.25 mu l of SAPE stock solution; 1×Tm hybridization solution (0.2M NaCl,0.1M Tris,0.08%Triton X-100, pH 8.0) 350. Mu.l. Immediately after removing the hybridization product from step 2) from the PCR apparatus, the supernatant was discarded after 60s on a magnetic plate (V & P Scientific, VP771LD-4 CS). And respectively adding 25 mu l of the prepared SAPE mixed solution into each reaction hole, sealing the film, and vibrating and uniformly mixing for 20s. The PCR instrument was set up and reacted as follows: 37℃for 15min. Immediately after the reaction, the 96-well plate was placed on a magnetic plate for 60s, and the supernatant was discarded. 50 μl of 1×Tm hybridization solution was added to each reaction well, followed by shaking and mixing for 20s, and then the supernatant was discarded after 30-60s in a magnetic plate, and repeated 1 time. Finally, 75 μl of 1×Tm hybridization solution was added to each reaction well, and mixed by shaking for 20s.

4) And (3) detecting a liquid-phase chip platform: and 3) detecting the product obtained in the step 3) by using a Luminex200 liquid-phase chip platform, wherein the detection operation steps and parameter setting are operated according to a Luminex200 operation instruction.

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

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

TABLE 4 multiple detection results of colorectal cancer samples of this example

In the detection results, the detection results of a plurality of sites of positive control are all more than 3 times of the corresponding NC value, and the method is proved to be capable of detecting a plurality of sites simultaneously; the samples of 5 patients all have at least 2 positive sites, and the control is negative, which proves that the method can better distinguish colorectal cancer samples.

Example 4 magnetic bead probe coupling

1. The probes used in this example were synthesized by the Kirschner Biotech company. Reagent MES (2- (N-morpholinoethanesulfonic acid) was purchased from Sigma Aldrich (Shanghai) trade Co., ltd. (Sigma M3671). EDAC N' - (ethyliminomethylene) -N, N-dimethyl-1, 3-propanediamine monohydrochloride was purchased from Sigma Aldrich (Shanghai) trade company, inc. (Sigma E7750). Tween-20 (Tween-20) was purchased from Beijing Soy Bao technology Co., ltd (Solarbio T8220) SDS (sodium dodecyl sulfate) was purchased from Beijing Soy Bao technology Co., ltd (Solarbio S8010). TE solution at pH 8.0: the composition of the material was 10mM Tris-HCl, 1mM EDTA.

The length of the probe sequence is 20-45bp, and the TM value is 75-85 DEG C

The probe sequence is as follows:

TZ-RUNX3 5'Amino Modifier C12-TAGCGGTCGTTAGGGCGTC G GGTAGGCGGAG-3'(SEQ ID NO.13)

the probe was diluted to 0.1nanomole with pure water for use.

4. Coupling step

3) 2. Mu.L of probe (0.1 nanomole) was added to the tube and vortexed.

Example 5 assay specific assay

1. The primers used in this example were synthesized by Kirschner, and plasmids containing the M-P16, M-RASSF1A, M-RUNX3, beta-ACTIN methylation site destination region and NC (methylation site-free destination region) were synthesized by Jin Weizhi Biotechnology Co., ltd; the reagents required for 2×Taq Master mix (the required Taq enzyme, dNTP mixture, mgCl2 and reaction buffer were pre-formulated as a 2-fold concentration mixture) were purchased from TAKALA company (TAKALA, RR 001B); SAPE stock was purchased from sameiser femoris technologies (china) limited (Life Technologies, S-866); TMAC (tetramethyl ammonium chloride solution) was purchased from Sigma aldrich (Shanghai) trade company (Sigma T3411); sarkosyl solution (sodium dodecyl sarcosinate solution) was purchased from Sigma aldrich (Shanghai) trade company (Sigma L7414); tris-HCl Tris (hydroxymethyl) aminomethane was purchased from beijing solibao technologies limited (Solarbio T8230); triton X-100 (polyethylene glycol p-isooctylphenyl ether) was purchased from Beijing Soy Bao technology Co., ltd (Solarbio T8200); EDTA (ethylenediamine tetraacetic acid) was purchased from Sairo BioFrox 1340GR 500; tris (Tris) is purchased from the Sairo BioFrox 1115GR 500); naCl (sodium chloride) was purchased from tianda chemical.

2. Primer probe design and synthesis

The PCR primer is designed aiming at methylation sites, the purity of the primer reaches electrophoresis grade (PAGE) or HPLC grade, the length of the primer sequence is 18-35bp, the TM value is 50-70 ℃, and the 5' end of the downstream primer is subjected to biotin modification.

An upstream primer: RUNX 3-M-F5'-ATAATAGCGGTCGTTAGGGCGTCG-3' (SEQ ID NO. 14)

A downstream primer: RUNX 3-M-R5 '-Biotin-GCTTCTACTTTCCCGCTTCTCGCG-3' (SEQ ID NO. 15)

Wherein Biotin represents a Biotin modification.

3. Synthesis of standard and negative reference

Dissolving dry powder of target region standard of M-P16, M-RASSF1A, M-RUNX3, M-beta-ACTIN methylation site and NC negative control standard in enzyme-free water, respectively, and diluting to 10 ¹⁵ Copy/ml, 10-fold gradient dilution of standard, 10 each ¹⁰ Copy/ml was used as a template for standard, and the specificity of RUNX3 magnetic bead-probe conjugates and RUNX3 primers were detected using M-P16, M-RASSF1A, M-RUNX3, M-beta-ACTIN, respectively.

4. Specificity analysis

1) M-P16, M-RASSF1A, M-RUNX3, M-beta-ACTIN methylation site target region standard, NC negative standard, and diluting according to the requirements to be used 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 5:

TABLE 5 10. Mu.l PCR amplification System in example 4 specificity analysis

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5. The detection results are shown in Table 6, M-P16, M-RASSF1A, M-RUNX3 and M-beta-ACTIN are target region standard products of methylation sites, and NC is a negative standard product.

TABLE 6 magnetic bead-probe conjugate specificity detection results

Therefore, the RUNX3 primer and the magnetic bead-probe conjugate of the detection method can specifically distinguish M-RUNX3 standard substances, which proves that the method has better specificity.

Example 6 multiplex detection assay

1. The primers and probes used in this example were synthesized by the gold srey biotechnology company; positive and negative standards were human methylated DNA and human unmethylated DNA (zymesearch, D5014) after bisulfite conversion, purchased from Zymo RESEARCH biology company (Zymo RESEARCH, D5014); tissue extraction kit was purchased from TIANGEN, DP304, a blood/cell/tissue genomic DNA extraction kit manufactured by TIANGEN biochemical technology (beijing) limited; 2 XTaq Master mix (Taq enzyme, dNTP mix desired; mgCl2 and reaction buffer pre-formulated as a 2-fold concentration mix) reagents were purchased from TAKALA company (TAKALA, RR 001B); the SAPE stock solution was purchased from sammer femto technology (china) limited (Life Technologies, S-866), and TMAC (tetramethyl ammonium chloride solution) was purchased from Sigma aldrich (Shanghai) trade limited (Sigma T3411); sarkosyl solution (sodium dodecyl sarcosinate solution) was purchased from Sigma aldrich (Shanghai) trade company (Sigma L7414); tris-HCl Tris (hydroxymethyl) aminomethane, purchased from beijing solebao technologies limited (Solarbio T8230); triton X-100 (polyethylene glycol p-isooctylphenyl ether) was purchased from Beijing Soy Bao technology Co., ltd (Solarbio T8200); EDTA (ethylenediamine tetraacetic acid) was purchased from Sairo BioFrox 1340GR 500; tris (Tris) is purchased from the Sairo BioFrox 1115GR 500); naCl (sodium chloride) was purchased from tianda chemical.

TZ-P16 5'Amino Modifier C12-GAGGGTGGGGCGGATCGCGTGCGTTCG-3'(SEQ ID NO.16)

TZ-β-actin 5'Amino Modifier C12TCGTGGTGGTGAAGTTGTAGTCGCGTTCGGTGAGG-3'(SEQ ID NO.5)

TZ-RASSF1A 5'Amino Modifier C12-TGGTATTCGTTGGGCG CGTTGGGAAG-3'(SEQ ID NO.17)。

an upstream primer: P16-M-F5'-ATTAGAGGGTGGGGCGGATCGC-3' (SEQ ID NO. 18)

Downstream primer P16-M-R5 'Biotin-ACCCCGAACCGCGACCGTAA-3' (SEQ ID NO. 19)

An upstream primer: RASSF 1A-M-F5'-GTTGGTATTCGTTGGGCGC-3' (SEQ ID NO. 20)

The downstream primer RASSF 1A-M-R5 'Biotin-AACTACCGTATAAAATTACACGCG-3' (SEQ ID NO. 21)

Internal reference gene primer:

an upstream primer: beta-ACTIN-M-F5'-TTTAATGTTACGTACGATTTTTC-3' (SEQ ID NO. 11)

Wherein Biotin represents a Biotin modification.

4. Extraction of tissue samples: DNA extraction kit (DP 304) for blood/cell/tissue genome DNA extraction from Tiangen Biochemical technology (Beijing) limited company, according to the method of the specification, DNA extraction was performed on 5 human gastric cancer tissue samples and 5 non-gastric cancer sample tissues.

6. Multiplex detection

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

TABLE 7 10. Mu.l PCR amplification System in multiplex detection example 6

7. The experimental results are shown in table 8, NC as a negative control and PC as a positive control.

TABLE 8 example 6 multiple gastric cancer sample detection results

Site(s)	Sample of	B39_RUNX3	B55_P16	B66_β-ACTIN	B72_RASSF1A
						1(1，A1)	C1	1682	107	1640	410.5
2(1，31)	C2	517.5	124	888	194
						3(1，C1)	C3	1875	1558.5	1348	371
4(1，D1)	C4	1736	149	1477	400.5
						5(1，E1)	C5	1446	406	1173	257
6(1，F1)	H1	81	112.5	422	101
						7(1，F1)	H2	90.5	89.5	867	112
8(1，G1)	H3	67	88	722	65
						9(1，A2)	H4	147.5	131	853.5	83
10(1，B2)	H5	90	172	1244	187.5
						11(1，C2)	PC	2070	1600	1994.5	1469
12(1，D2)	NC	73.5	78	76	60
						13(1，E2)	H	79	88.5	84	68

In the detection results, the detection results of a plurality of sites of positive control are all more than 3 times of the corresponding NC value, and the method is proved to be capable of detecting a plurality of sites simultaneously; the samples of 5 patients all have at least 2 positive sites, and the control is negative, which proves that the method can better distinguish gastric cancer samples.

Example 7 magnetic bead probe coupling

1. The probes used in this example were synthesized by the Kirschner Biotech company. Reagent MES (2- (N-morpholinoethanesulfonic acid) was purchased from Sigma Aldrich (Shanghai) trade Co., ltd. (Sigma M3671). EDACN' - (ethyliminomethylene) -N, N-dimethyl-1, 3-propanediamine monohydrochloride was purchased from Sigma Aldrich (Shanghai) trade company, inc. (Sigma E7750). Tween-20 (Tween-20) was purchased from Beijing Soy Bao technology Co., ltd (Solarbio T8220). SDS (sodium dodecyl sulfate) was purchased from Beijing Soy Bao technology Co., ltd (Solarbio S8010). TE solution at pH 8.0: the composition of the material was 10mM Tris-HCl, 1mM EDTA.

The length of the probe sequence is 20-45bp, and the TM value is 75-90 DEG C

The probe sequence is as follows:

NPTX2 5'Amino Modifier C12-CGCGATCGGTGCGGTTGTGAGACGGTGATCGCG-3'(SEQ ID NO.22)

the probe was diluted to 0.1nanomole with pure water for use.

4. Coupling step

3) 2. Mu.L of probe (0.1 nanomole) was added to the tube and vortexed.

Example 8 assay specific assay

1. The primers used in this example were synthesized by Kirschner, inc., and plasmids containing the target region of the methylation site of M-APC, M-NPTX2,. Beta. -ACTIN and NC (target region without methylation site) were synthesized by Jin Weizhi Biotech Co., ltd; the reagents required for 2×Taq Master mix (the required Taq enzyme, dNTP mixture, mgCl2 and reaction buffer were pre-formulated as a 2-fold concentration mixture) were purchased from TAKALA company (TAKALA, RR 001B); SAPE stock was purchased from sameiser femoris technologies (china) limited (Life Technologies, S-866); TMAC (tetramethyl ammonium chloride solution) was purchased from Sigma aldrich (Shanghai) trade company (Sigma T3411); sarkosyl solution (sodium dodecyl sarcosinate solution) was purchased from Sigma aldrich (Shanghai) trade company (Sigma L7414); tris-HCl Tris (hydroxymethyl) aminomethane was purchased from beijing solibao technologies limited (Solarbio T8230); triton X-100 (polyethylene glycol p-isooctylphenyl ether) was purchased from Beijing Soy Bao technology Co., ltd (Solarbio T8200); EDTA (ethylenediamine tetraacetic acid) was purchased from Sairo BioFrox 1340GR 500; tris (Tris) is purchased from the Sairo BioFrox 1115GR 500); naCl (sodium chloride) was purchased from tianda chemical. TE solution at pH 8.0: the composition of the material was 10mM Tris-HCl, 1mM EDTA.

2. Primer probe design and synthesis

The PCR primer is designed aiming at methylation sites, the purity of the primer reaches electrophoresis grade (PAGE) or HPLC grade, the length of the primer sequence is 15-30bp, the TM value is 50-65 ℃, and the 5' -end of the downstream primer is subjected to biotin modification.

An upstream primer: NPTX 2-M-F5'-TTCGGTAGGTTAGAGTGT C-3' (SEQ ID NO. 23)

A downstream primer: NPTX 2-M-R5 '-Biotin-CTATCGTCTCGAAAATCGCGC-3' (SEQ ID NO. 27)

Wherein Biotin represents a Biotin modification.

3. Synthesis of standard and negative reference

Dissolving dry powder of target region standard of M-APC, M-NPTX2, beta-ACTIN methylation site and NC negative control standard with enzyme-free water, respectively, and diluting to 10 ¹⁵ Copy/ml, 10-fold gradient dilution of standard, 10 each ¹⁰ Copy/ml was used as a template for the standard, using M-APC, M-NPTX2, beta-ACTIN to detect the specificity of NPTX2 magnetic bead-probe conjugate and NPTX2 primer, respectively.

4. Specificity analysis

1) M-APC, M-NPTX2, beta-ACTIN methylation site target region standard, NC negative standard, are diluted according to the requirements and used as templates to be detected, and the detection is carried out according to the following steps:

2) The PCR amplification system of this example is shown in Table 9:

TABLE 9 10. Mu.l PCR amplification System in example 9 specificity analysis

5. The detection results are shown in Table 10, M-APC, M-NPTX2, beta-ACTIN is the target region standard of methylation site, NC is the negative standard.

TABLE 10 magnetic bead-probe conjugate specificity detection results

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Therefore, the NPTX2 primer and the magnetic bead-probe conjugate of the detection method can specifically distinguish NPTX2 standard substances, which proves that the method has better specificity.

Example 9 multiplex detection analysis

1. The primers and probes used in this example were synthesized by the gold srey biotechnology company; the negative standard was human unmethylated DNA transformed with bisulfite, purchased from Zymo RESEARCH biology company (Zymo RESEARCH, D5014); blood DNA samples were extracted from QIAGEN company (QIAGEN, 55114); 2 XTaq Master mix (Taq enzyme, dNTP mix desired; mgCl2 and reaction buffer pre-formulated as a 2-fold concentration mix) reagents were purchased from TAKALA company (TAKALA, RR 001B); the SAPE stock solution was purchased from sammer femto technology (china) limited (Life Technologies, S-866), and TMAC (tetramethyl ammonium chloride solution) was purchased from Sigma aldrich (Shanghai) trade limited (Sigma T3411); sarkosyl solution (sodium dodecyl sarcosinate solution) was purchased from Sigma aldrich (Shanghai) trade company (Sigma L7414); tris-HCl Tris (hydroxymethyl) aminomethane, purchased from beijing solebao technologies limited (Solarbio T8230); triton X-100 (polyethylene glycol p-isooctylphenyl ether) was purchased from Beijing Soy Bao technology Co., ltd (Solarbio T8200); EDTA (ethylenediamine tetraacetic acid) was purchased from Sairo BioFrox 1340GR 500; tris (Tris) is purchased from the Sairo BioFrox 1115GR 500); naCl (sodium chloride) was purchased from tianda chemical.

TZ-APC 5'Amino Modifier C12-CGCGATCGTTGGATGCGGAATCGCG-3'(SEQ ID NO.24)

TZ-β-actin 5'Amino Modifier C12-TCGTGGTGGTGAAGTTGTAGTCGCGTTCGGTGAGG-3'(SEQ ID NO.5)

an upstream primer: APC-M-F5'-ATTGCGGAGTGCGGGTC-3' (SEQ ID NO. 25)

The downstream primer is APC-M-R5 'Biotin-AATCGACGAACTCCCGACG-3' (SEQ ID NO. 26)

Internal reference gene primer:

an upstream primer: beta-ACTIN-M-F5'-TTTAATGTTACGTACGATTTTTC-3' (SEQ ID NO. 11)

Wherein Biotin represents a Biotin modification.

4. Extraction of blood samples: free nucleic acid DNA extraction kit (55114) manufactured by Qiagen, inc., DNA extraction was performed on 3 pancreatic cancer samples and 5 healthy blood samples according to the method of the specification

6. Multiplex detection

1) The PCR amplification system of this example is shown in Table 11 below:

TABLE 11 10 μl PCR amplification System in example 9 multiplex detection

7. The experimental results are shown in table 12, NC being a negative control and PC being a positive control.

TABLE 12 multiple detection results of pancreatic cancer samples of this example 9

In the detection results, the detection results of a plurality of sites of positive control are all more than 3 times of the corresponding NC value, and the method is proved to be capable of detecting a plurality of sites simultaneously; the method has the advantages that no less than 1 site in 3 patient samples is positive, and the control is negative, so that pancreatic cancer samples can be well distinguished.

The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Numerous variations of the presently illustrated embodiments of the application will be apparent to those of ordinary skill in the art and are intended to be within the scope of the appended claims and equivalents thereof.

Sequence listing

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Claims

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

c) Detecting the signal of the hybridization product.

2. The method of claim 1, wherein the gene of interest is selected from the group consisting of:

a) SEPT9, FOXE1, and VIM;

b) RUNX3, P16 and RASSFIA; and, a step of, in the first embodiment,

c) APC and NPTX2.

3. The method of any one of claims 1-2, wherein the downstream primer comprises a nucleotide sequence set forth in any one of SEQ ID nos. 3,8, 10,15,19,21,26,27.

4. A method according to any one of claims 1 to 3, wherein the upstream primer comprises a nucleotide sequence set forth in any one of SEQ ID No.2,7,9,14,18,20,23,25.

5. The method of any one of claims 1-4, wherein the probe comprises a nucleotide sequence set forth in any one of SEQ ID nos. 1,4,6,13,16,17,24.

6. The method of any one of claims 1-5, wherein the carrier is linked to the probe through the amino-modifying functional group.

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

8. The method of any one of claims 1-7, wherein the number of PCR amplifications of step b) is 1.

9. The method according to any one of claims 1-8, wherein the method further comprises the steps of:

c) Detecting the signal of the hybridization product.

10. A primer pair comprising the upstream primer of any one of claims 1-9 and the downstream primer of any one of claims 1-9.