WO2022143396A1

WO2022143396A1 - Reagent kit for methylation detection of breast tumor specificity

Info

Publication number: WO2022143396A1
Application number: PCT/CN2021/140808
Authority: WO
Inventors: 赵德志; 杨婷; 王军; 张显玉; 庞达; 陈志伟; 范建兵
Original assignee: 广州市基准医疗有限责任公司
Priority date: 2020-12-28
Filing date: 2021-12-23
Publication date: 2022-07-07
Also published as: CN112646888B; CN112646888A

Abstract

Provided is a detection reagent kit for a methylation biomarker related to a breast cancer, comprising for a biomarker cg23035715, a primer and a probe selected from any one group from the following groups: SEQ ID NO.1-SEQ ID NO:3, SEQ ID NO.70-SEQ ID NO:72, and SEQ ID NO.139-SEQ ID NO:141.

Description

Breast tumor-specific methylation detection kit

technical field

The invention belongs to the field of biotechnology, and in particular relates to a kit for breast tumor-specific methylation detection.

Background technique

According to the China Cancer Registry Annual Report, the incidence of female breast cancer is at a low level in the age group of 0-24 years old, gradually increases after the age of 25, reaches a peak in the age group of 50-54 years old, and gradually decreases after the age of 55. European and American countries have done a lot of research on breast cancer susceptibility genes. Now known are BRCA-1, BRCA-2, p53, PTEN, etc. Breast cancer related to these gene mutations is called hereditary breast cancer Cancer, accounting for 5% to 10% of all breast cancers. The breast cancer 21-gene test not only provides prediction of recurrence risk after 1-5 years and after 5 years, but also serves as the only multi-gene test to predict the benefit of chemotherapy and endocrine therapy in patients with estrogen receptor-positive invasive breast cancer. Breast cancer 70 gene testing also plays an important role in early breast cancer diagnosis. The latest research finds 72 new common genetic variants that increase the risk of breast cancer in women, bringing the number of gene mutations known to be associated with breast cancer to 177. Many people may carry common genetic variants, and a single gene mutation has a relatively small risk of cancer, but the more related variants in a woman's body, the greater the risk of breast cancer. The current breast cancer genetic testing method is a non-invasive test, but it needs to be based on the test of the tissue removed during the original surgery (tumorectomy, mastectomy or core needle biopsy).

Early screening and diagnosis of tumors can detect early-stage cancer with a higher probability, thereby improving its five-year survival rate and reducing mortality. Current status of breast cancer screening in China: 5-year survival rate of breast cancer: 100% for carcinoma in situ, 84-100% for stage I, 76-87% for stage II, and 38-77% for stage III. According to a Chinese multicenter study, at the time of initial diagnosis of breast cancer, 15.7% stage I, 44.9% stage II, 18.7% stage III, and 2.4% stage IV. Breast cancer imaging screening methods: 1. Mammographic target X-ray: Chinese women are younger (45-55 years old), breast glands are denser than Western women, MG is easy to miss, and MG is sensitive to the diagnosis of dense breast lesions Only 30%. 2. B-ultrasound: the positive predictive value of MG alone was 55.5%, and the positive predictive value of MG combined with B-ultrasound was 43.3%. 3. Magnetic resonance imaging (MRI): MRI is not sensitive to small calcifications and has clear contraindications; therefore, early screening and early treatment of tumors is of great significance to improve the five-year survival rate. The key is to establish an effective detection model and find molecular markers that can be used as screening and diagnosis.

cfDNA (cell-free DNA) is a small fragment of free nucleic acid DNA in peripheral blood, derived from the metabolism and apoptosis of normal cells or tumor cells, and contains genetic information such as somatic mutation and DNA methylation. By detecting disease-specific cfDNA fragments, the technology of mastering the occurrence and development of diseases is called liquid biopsy (Liquid Biopsy). Compared with traditional tissue biopsy, it has many advantages such as rapidity, convenience and less damage. In 2015, Professor Lu Yuming proved the technical and theoretical feasibility of liquid biopsy to replace tissue biopsy through cfDNA whole-genome methylation sequencing; in 2017, Professor Zhang Kun's team used ctDNA methylation to quantitatively describe tumor burden and tumor origin. ctDNA profile. More and more clinical studies have shown that plasma ctDNA can be used as a biomarker for early diagnosis and screening of tumors, prediction, response to treatment, and monitoring tumor size and recurrence. At present, the international research direction is to integrate multiple omics, multiple molecular markers, multiple genes, and multiple loci to improve the sensitivity and specificity of detection technology to meet clinical needs for detection products.

DNA methylation is an epigenetic modification of genes. Compared with cancer patients at different stages and healthy individuals, there are significant differences in DNA methylation patterns and methylation levels. There will be slight changes in the level of DNA methylation. Therefore, accurate and quantitative detection of DNA methylation markers is of great significance for the early diagnosis of cancer.

In an earlier study, the inventors of the present invention screened out methylation biomarkers or their combination that can be used for breast cancer detection (see CN2020104264964), by studying the genomic fragments of these plasma cfDNA methylation modification patterns in breast cancer at various stages Differences in methylation modification between cancer patients and healthy people, and found that the combination of the above plasma genome fragments can be used as early diagnosis markers for breast cancer. Moreover, the inventors found that one of the methylation markers (cg23035715) or in combination with the first three methylation markers (cg26371731, cg04541368, cg13973436) all possessed 26 overlapped with those screened by the two algorithms LASSO and Random Forest. The diagnostic power of the combination of the methylation markers was similar.

MethyLight fluorescence quantification utilizes TaqMan probes and PCR primers to differentiate between methylated and unmethylated DNA. The DNA fragments are first treated with sulfite, and a probe is designed for methylation and unmethylation, labeled with different fluorophores, followed by real-time quantitative PCR. A fluorescent signal is released if the probe hybridizes to DNA, and the methylation level is calculated from the ratio of the fluorescent signals. This method is suitable for studies with large sample size but few sites.

However, at present, the detection of polygenes in plasma cfDNA methylation of breast tumor patients has high requirements for low investment in library construction due to the small amount of cfDNA, and low investment and damage affect the final bioinformatics analysis, and the sensitivity requirements for the entire assay high defect.

SUMMARY OF THE INVENTION

One of the objectives of the present invention is to provide a high-sensitivity test kit for breast cancer-related methylation biomarkers, which combines multiplex PCR technology to target enrichment of multiple genes and MethLight technology, and first optimizes the multiplex PCR , high specificity targeted enrichment, and then through specific screening of primers and probes, single-gene detection has high sensitivity and specificity.

The technical solution to achieve the above object is as follows.

A detection kit for breast cancer-related methylation biomarkers, comprising: primers and probes selected from any of the following groups for biomarker cg23035715: SEQ ID NO.1-SEQ ID NO.3, SEQ ID NO.70-SEQ ID NO.72, SEQ ID NO.139-SEQ ID NO.141.

In one embodiment, it also includes primers and probes for at least one biomarker of cg26371731, cg04541368 and cg13973436; the primers and probes for cg26371731 biomarkers are selected from: SEQ ID NO.4- SEQ ID NO.6, SEQ ID NO.73-SEQ ID NO.75, SEQ ID NO.142-SEQ ID NO.144;

The primers and probes for the cg04541368 biomarker are selected from: SEQ ID NO.7-SEQ ID NO.9, SEQ ID NO.76-SEQ ID NO.78, SEQ ID NO.145-SEQ ID NO.147 ;

The primers and probes for the cg13973436 biomarker are selected from: SEQ ID NO.10-SEQ ID NO.12, SEQ ID NO.79-SEQ ID NO.81, SEQ ID NO.148-SEQ ID NO.150 .

In one embodiment, it also includes primers and probes selected from cg26371731, cg04541368 and cg13973436 biomarkers; the primers and probes for cg26371731 biomarkers are: SEQ ID NO.4-SEQ ID NO. 6; the primers and probes for the cg04541368 biomarker are: SEQ ID NO.7-SEQ ID NO.9;

The primers and probes for the cg13973436 biomarker are: SEQ ID NO.10-SEQ ID NO.12.

在其中一个实施例中，还包括选自cg16304215，cg20072171，cg21501525，cg22778178，cg08599259，cg25566568，cg15634980，cg07458308，cg01348584，cg14140881，cg25756435，cg00594560，cg18087672，cg14868703，cg17632299，cg18786873，cg20631750，cg25924096，和cg15321298中Primers and probes for at least one biomarker;

The primers and probes for the cg16304215 biomarker are selected from: SEQ ID NO.13-SEQ ID NO.15, SEQ ID NO.82-SEQ ID NO.84, SEQ ID NO.151-SEQ ID NO.153 ;

The primers and probes for the cg20072171 biomarker are selected from: SEQ ID NO.16-SEQ ID NO.18, SEQ ID NO.85-SEQ ID NO.87, SEQ ID NO.154-SEQ ID NO.156 ;

The primers and probes for the cg21501525 biomarker are selected from: SEQ ID NO.19-SEQ ID NO.21, SEQ ID NO.88-SEQ ID NO.90, SEQ ID NO.157-SEQ ID NO.159 ;

The primers and probes for the cg22778178 biomarker are selected from: SEQ ID NO.22-SEQ ID NO.24, SEQ ID NO.91-SEQ ID NO.93, SEQ ID NO.160-SEQ ID NO.162 ;

The primers and probes for the cg08599259 biomarker are selected from: SEQ ID NO.25-SEQ ID NO.27, SEQ ID NO.94-SEQ ID NO.96, SEQ ID NO.163-SEQ ID NO.165 ;

The primers and probes for the cg25566568 biomarker are selected from: SEQ ID NO.28-SEQ ID NO.30, SEQ ID NO.97-SEQ ID NO.99, SEQ ID NO.166-SEQ ID NO.168 ;

The primers and probes for the cg15634980 biomarker are selected from: SEQ ID NO.31-SEQ ID NO.33, SEQ ID NO.100-SEQ ID NO.102, SEQ ID NO.169-SEQ ID NO.171 ;

The primers and probes for the cg07458308 biomarker are selected from: SEQ ID NO.34-SEQ ID NO.36, SEQ ID NO.103-SEQ ID NO.105, SEQ ID NO.172-SEQ ID NO.174 ;

The primers and probes for the cg01348584 biomarker are selected from: SEQ ID NO.37-SEQ ID NO.39, SEQ ID NO.106-SEQ ID NO.108, SEQ ID NO.175-SEQ ID NO.177 ;

The primers and probes for the cg14140881 biomarker are selected from: SEQ ID NO.40-SEQ ID NO.42, SEQ ID NO.109-SEQ ID NO.111, SEQ ID NO.178-SEQ ID NO.180 ;

The primers and probes for the cg25756435 biomarker are selected from: SEQ ID NO.43-SEQ ID NO.45, SEQ ID NO.112-SEQ ID NO.114, SEQ ID NO.181-SEQ ID NO.183 ;

The primers and probes for the cg00594560 biomarker are selected from: SEQ ID NO.46-SEQ ID NO.48, SEQ ID NO.115-SEQ ID NO.117, SEQ ID NO.184-SEQ ID NO.186 ;

The primers and probes for the cg18087672 biomarker are selected from: SEQ ID NO.49-SEQ ID NO.51, SEQ ID NO.118-SEQ ID NO.120, SEQ ID NO.187-SEQ ID NO.189 ;

The primers and probes for the cg14868703 biomarker are selected from: SEQ ID NO.52-SEQ ID NO.54, SEQ ID NO.121-SEQ ID NO.123, SEQ ID NO.190-SEQ ID NO.192 ;

The primers and probes for the cg17632299 biomarker are selected from: SEQ ID NO.55-SEQ ID NO.57, SEQ ID NO.124-SEQ ID NO.126, SEQ ID NO.193-SEQ ID NO.195 ;

The primers and probes for the cg18786873 biomarker are selected from: SEQ ID NO.58-SEQ ID NO.60, SEQ ID NO.127-SEQ ID NO.129, SEQ ID NO.196-SEQ ID NO.198 ;

The primers and probes for the cg20631750 biomarker are selected from: SEQ ID NO.61-SEQ ID NO.63, SEQ ID NO.130-SEQ ID NO.132, SEQ ID NO.199-SEQ ID NO.201 ;

The primers and probes for the cg25924096 biomarker are selected from: SEQ ID NO.64-SEQ ID NO.66, SEQ ID NO.133-SEQ ID NO.135, SEQ ID NO.202-SEQ ID NO.204 ;

The primers and probes for the cg15321298 biomarker are selected from: SEQ ID NO.67-SEQ ID NO.69, SEQ ID NO.136-SEQ ID NO.138, SEQ ID NO.205-SEQ ID NO.207 .

In one embodiment, primers and probes for the internal reference gene are also included, and the primers and probes for the internal reference gene are: SEQ ID NO.208-SEQ ID NO.210.

在其中一个实施例中，还包括选自cg16304215，cg20072171，cg21501525，cg22778178，cg08599259，cg25566568，cg15634980，cg07458308，cg01348584，cg14140881，cg25756435，cg00594560，cg18087672，cg14868703，cg17632299，cg18786873，cg20631750，cg25924096，和cg15321298的Primers and Probes.

In one embodiment, the primers and probes for cg16304215 biomarkers are SEQ ID NO.13-SEQ ID NO.15; the primers and probes for cg20072171 biomarkers are SEQ ID NO.16 -SEQ ID NO.18;

The primers and probes for the cg21501525 biomarker are SEQ ID NO.19-SEQ ID NO.21;

The primers and probes for the cg22778178 biomarker are SEQ ID NO.22-SEQ ID NO.24;

The primers and probes for the cg08599259 biomarker are SEQ ID NO.25-SEQ ID NO.27;

The primers and probes for the cg25566568 biomarker are SEQ ID NO.28-SEQ ID NO.30;

The primers and probes for the cg15634980 biomarker are SEQ ID NO.31-SEQ ID NO.33;

The primers and probes for the cg07458308 biomarker are SEQ ID NO.34-SEQ ID NO.36;

The primers and probes for the cg01348584 biomarker are SEQ ID NO.37-SEQ ID NO.39;

The primers and probes for the cg14140881 biomarker are SEQ ID NO.40-SEQ ID NO.42;

The primers and probes for the cg25756435 biomarker are SEQ ID NO.43-SEQ ID NO.45;

The primers and probes for the cg00594560 biomarker are SEQ ID NO.46-SEQ ID NO.48;

The primers and probes for the cg18087672 biomarker are SEQ ID NO.49-SEQ ID NO.51;

The primers and probes for the cg14868703 biomarker are SEQ ID NO.52-SEQ ID NO.54;

The primers and probes for the cg17632299 biomarker are SEQ ID NO.55-SEQ ID NO.57;

The primers and probes for the cg18786873 biomarker are SEQ ID NO.58-SEQ ID NO.60;

The primers and probes for the cg20631750 biomarker are SEQ ID NO.61-SEQ ID NO.63;

The primers and probes for the cg25924096 biomarker are SEQ ID NO.64-SEQ ID NO.66;

The primers and probes for the cg15321298 biomarker are SEQ ID NO.67-SEQ ID NO.69.

In one embodiment, a PCR reaction solution is also included, and the PCR reaction solution includes DNATaq polymerase, dNTPs, Mg ²⁺ and 10×DNA polymerase buffer.

In one embodiment, the sample to be detected by the detection kit is blood, serum or plasma or a tissue sample.

In one embodiment, a primer mixture is also included. In the amplification system, the concentration of each primer is 300±10nM; the concentration of magnesium ions is 1.5±0.1mM, and the concentration of dNTP mixture is 400±10uM; the reaction enzyme Phusion U, primer-probe mixture, in multiplex fluorescence quantitative PCR reaction: each primer concentration is 900±10nM; probe concentration is 200±10nM.

The invention firstly uses the unique multiplex PCR technology to target and enrich the multi-gene targeted region, avoiding the problem of low original cfDNA in multi-gene detection, and by combining the low cost and rapidity of MethyLight PCR detection, the detection throughput is improved, and at the same time The sensitivity and specificity of model detection are also improved. The detection result of the primer-probe of the single molecular marker cg16304215 has good sensitivity, and its combination with the primer-probe of any one of the other molecular markers has stronger and more stable diagnostic ability. The detection kit of the present invention can well improve the sensitivity and specificity of breast cancer detection, effectively improve the detection rate of early breast cancer, perform early treatment and intervention, and improve the survival rate of patients.

The fluorescent quantitative PCR detection primers and probes for the DNA methylation molecular markers provided by the kit of the present invention overcome the disadvantage of mutual interference when multiple primers and probes perform multiple PCR amplification and detection. When using the primers to perform multiplex PCR on bisulfite-treated DNA, each DNA methylation molecular marker can be effectively amplified and enriched; when multiple PCR products are subsequently detected by multiplex fluorescence quantitative PCR, The _{CT value obtained by the corresponding DNA methylation molecular marker in the multiplex fluorescence quantitative PCR detection is not significantly different from the CT} _value of the DNA methylation molecular marker alone in the fluorescent quantitative PCR reaction, and the quantitative performance is equivalent to that of a single region. . The kit is optimized so that the primers and probes for different DNA methylation molecular markers do not interfere with each other, and can successfully realize multiple PCR amplification and multiple fluorescence quantitative PCR detection, effectively improving the detection efficiency.

The detection method for the DNA methylation molecular marker provided by the present invention can effectively enrich the target molecule by introducing multiple PCR amplification, overcome the limitation of low sample acquisition, and also amplify the detection signal while also amplifying the detection signal. The combined detection of multiple molecular markers can improve the detection sensitivity and detection efficiency, and can enhance the detection rate of breast cancer.

Description of drawings

Figure 1 shows the combined ROC curve of methylation markers for 1 marker (cg23035715) and 4 markers (cg23035715, cg26371731, cg04541368 and cg13973436) in the independent validation set of sequencing methods.

Figure 2 shows the sensitivity and specificity of different breast cancer stages in random combinations of cg23035715, cg26371731, cg04541368 and cg13973436 methylation markers in the sequencing-independent validation set.

Figure 3 is the ROC curve (a) of the cg23035715 marker in the independent validation set of the sequencing method and the randomly selected one marker cg25924096 (a) and the sensitivity and specificity (b) of different breast cancer stages.

FIG. 4 is the amplification curve of one of the molecular markers (Marke9) in Example 3 detected by fluorescence quantitative PCR reaction.

5 is the ROC curve of the marker combinations A(a), B(b) and K(c) in Example 4. FIG.

6 is the ROC curve of the marker combinations A/B(a), A/C/J(b), A/B/C/J(c) and A-L(d) in Example 4. FIG.

Detailed ways

The experimental methods of unreceipted specific conditions in the following examples of the present invention are usually in accordance with conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York:Cold Spring Harbor Laboratory Press, 1989), or Follow the conditions recommended by the manufacturer. Various common chemical reagents used in the examples are all commercially available products.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used in the description of the present invention are only for the purpose of describing specific embodiments, and are not used to limit the present invention.

The terms "comprising" and "having" and any variations thereof of the present invention are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, product or device comprising a series of steps is not limited to the listed steps or modules, but optionally also includes unlisted steps, or optionally also includes steps for these processes, other steps inherent in the method, product or device.

The "plurality" mentioned in the present invention means two or more. "And/or", which describes the association relationship of the associated objects, means that there can be three kinds of relationships, for example, A and/or B, which can mean that A exists alone, A and B exist at the same time, and B exists alone. The character "/" generally indicates that the associated objects are an "or" relationship.

Unless specifically stated or otherwise defined, "first, second..." used herein is only used to distinguish names, and does not represent a specific number or order.

In order to facilitate understanding of the present invention, the present invention will be described more fully below. The present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough and complete understanding of the present disclosure will be provided.

Table 1 26 methylation markers

生物标记物biomarker	chromsomechromsome	hg19_starthg19_start	hg19_endhg19_end	GeneGene	strandstrand	SEQ ID NO.SEQ ID NO.
cg23035715cg23035715	chr1chr1	223302410223302410	223303011223303011	BMP7BMP7	++	211211
cg26371731cg26371731	chr1chr1	156389940156389940	156390541156390541	C1orf61C1orf61	++	212212
cg04541368cg04541368	chr5chr5	9290912992909129	9290973092909730	NR2F1-AS1NR2F1-AS1	++	213213
cg13973436cg13973436	chr16chr16	6742859867428598	6742919967429199	ZDHHC1ZDHHC1	++	214214
cg16304215cg16304215	chr5chr5	7692851076928510	7692911176929111	OTPOTP	++	215215
cg20072171cg20072171	chr3chr3	6235666162356661	6235726262357262	FEZF2FEZF2	--	216216
cg08402365cg08402365	chr2chr2	172944973172944973	172945574172945574	METAP1DMETAP1D	--	217217
cg21501525cg21501525	chr19chr19	3184364731843647	3184424831844248	TSHZ3TSHZ3	--	218218
cg22778178cg22778178	chr17chr17	4804165548041655	4804225648042256	FLJ45513FLJ45513	--	219219
cg08599259cg08599259	chr1chr1	3851063238510632	3851123338511233	POU3F1POU3F1	--	220220
cg25566568cg25566568	chr14chr14	3699470936994709	3699531036995310	NKX2-1-AS1NKX2-1-AS1	++	221221

cg15634980cg15634980	chr6chr6	3625272636252726	3625332736253327	PNPLA1PNPLA1	++	222222
cg07458308cg07458308	chr5chr5	134827211134827211	134827812134827812	TIFABTIFAB	--	223223
cg01348584cg01348584	chr7chr7	49107224910722	49113234911323	RADILRADIL	++	224224
cg14140881cg14140881	chr7chr7	105221719105221719	105222320105222320	EFCAB10EFCAB10	++	225225
cg25756435cg25756435	chr19chr19	1295182812951828	1295242912952429	MAST1MAST1	--	226226
cg00594560cg00594560	chr8chr8	7759294177592941	7759354277593542	ZFHX4-AS1ZFHX4-AS1	--	227227
cg08279008cg08279008	chr2chr2	8626292486262924	8626352586263525	POLR1APOLR1A	++	228228
cg09760908cg09760908	chr2chr2	4502841245028412	4502901345029013	CAMKMTCAMKMT	++	229229
cg18087672cg18087672	chr17chr17	4682461546824615	4682521646825216	HOXB13HOXB13	++	230230
cg14868703cg14868703	chr12chr12	113909647113909647	113910248113910248	LHX5-AS1LHX5-AS1	--	231231
cg17632299cg17632299	chr13chr13	5331266653312666	5331326753313267	LECT1LECT1	++	232232
cg18786873cg18786873	chr1chr1	110610598110610598	110611199110611199	ALX3ALX3	--	233233
cg20631750cg20631750	chr20chr20	5583926355839263	5583986455839864	BMP7BMP7	--	234234
cg25924096cg25924096	chr6chr6	100054285100054285	100054886100054886	PRDM13PRDM13	++	235235
cg15321298cg15321298	chr6chr6	150311425150311425	150312026150312026	ULBP1ULBP1	--	236236

In the previous research, the inventors used the information of 26 methylation markers (SEQ ID NO. Among them, 3 cases in

stage

0, 12 cases in stage I, 22 cases in stage II, 8 cases in stage III, and 2 cases in stage IV) were subjected to random combination test. Carry out marker screening and model establishment (refer to previous research CN2020104264964), use logistic regression model for modeling analysis, and further screen 4 markers (cg23035715, cg26371731, cg04541368 and cg13973436), specific data analysis methods and Example 2 Consistently, Top1 (cg23035715) and Top1/2/3/4 (cg23035715, cg26371731, cg04541368, and cg13973436) had 98% specificity (22/22), and the detection sensitivity of breast cancer stage 0 in the validation set was 100%, respectively (3/3) and 100% (3/3), the detection sensitivity of breast cancer stage I was 91.67% (11/12) and 91.67% (11/12), respectively, and the detection sensitivity of breast cancer stage II was 90.90% (20/22) and 90.91% (20/22), the detection sensitivity of stage III was 87.5% (7/8) and 87.5% (7/8), respectively, and the detection sensitivity of stage IV breast cancer was 50% (1 /2) and 0% (0/2), for all breast cancer samples, the overall sensitivity of the detection was 91.49% (43/47), 87.23% (41/47), and the overall AUC was 0.9395 and 0.9796, respectively, indicating that These loci combinations are highly correlated with the early diagnosis of breast cancer. See Figure 1 and Figure 2a-2b for the results.

The 4 biomarkers: cg23035715, cg26371731, cg04541368 and cg13973436, wherein the AUC of the single marker cg23035715 is 0.9395, see Figure 1, and Figure 1 shows 4 markers (cg23035715, The combination of cg26371731, cg04541368 and cg13973436) compared with marker1 (cg23035715) markers in the ability to identify breast cancer, as can be seen from Figure 1, the AUC of the combination of 4 methylation markers is already very close to 26 markers The combined AUC of the marker1 marker was also 0.9395, indicating the importance of these four methylation markers, especially the marker1 marker has a strong ability to diagnose early breast cancer.

As shown in Figure 2c-2d, at 98% specificity, the detection sensitivity of Top1/2/3 and Top1/2/4 methylation marker combinations in phase 0 of the validation set was 100% (3/3), respectively and 33.33% (1/3), the detection sensitivity of phase I was 100% (12/12) and 66.67% (8/12), and the detection sensitivity of phase II was 81.82% (18/22) and 63.63% ( 14/22), the detection sensitivity of stage III was 87.5% (7/8) and 75% (6/8), and the detection sensitivity of stage IV was 50% (1/2) and 0% (0/2), respectively, The overall sensitivity of the overall detection was 85.11% (40/47) and 61.7% (29/47), respectively. Among them, top1/2/3 in Figure 2c represents the combination of cg23035715, cg26371731 and cg04541368, while top1/2/4 represents the combination of the three markers (cg23035715, cg26371731 and cg13973436), and so on.

Further validation of the importance of top1 alone, at 98% specificity, top1 (SEQ ID NO. 1) in combination with cg23035715, cg16304215 and cg08599259 in random SEQ ID NO. 5-26 markers and with the remaining 40 markers (SEQ ID NO. 27-66) randomly selected combinations of cg01167274, cg07790615, cg23134869 and cg01832036, the detection sensitivities of phase 0 of the validation set were 100% (3/3) and 100% (3/3), respectively. The detection sensitivity of phase II was 100% (12/12) and 83.33% (10/12), respectively, the detection sensitivity of phase II was 95.45% (21/22) and 95.45% (21/22), and the detection sensitivity of phase III were 100% (8/8) and 87.5% (7/8), respectively, the stage IV detection sensitivity was 0% (0/2) and 0% (0/2), and the overall sensitivity of the overall detection was 93.61% ( 40/47) and 61.7% (29/47), see Figures 3a-3b and 3a-3b.

According to the results of the above-mentioned 22 normal people with no abnormalities in mammography and color Doppler ultrasound and 47 patients with breast cancer plasma samples, cg23035715 has a good performance in the diagnosis of early breast cancer, and the combination with cg26371731, cg04541368 and cg13973436 also showed The diagnostic ability was similar to the combination of top1-26, and cg23035715 was combined with cg23035715, cg16304215 and cg08599259 in random SEQ ID NO.5-26 markers, and the AUC of top1/2/3/4 markers was also above 0.75, For example, the AUCs of cg2303571, cg26371731, cg04541368 and cg13973436 were 0.9395, 0.8020, 0.7701 and 0.7501, as shown in Table 2, indicating that the loci screened by this method had a very high correlation with the early diagnosis of breast cancer.

Table 2. Performance of 26 methylation marker fragments in breast cancer diagnosis - AUC.

The invention discloses primers and probes and a combined PCR reaction system for detecting methylation markers (23 types) for breast cancer diagnosis. The breast cancer-specific methylation markers were strictly screened according to the TCGA database, and 23 breast cancer plasma markers were screened out of the original 26 biomarkers through targeted methylation high-throughput screening, combined with methylation-specific polymerase. Chain reaction enrichment and MethyLight PCR fluorescence were used to detect methylation markers of multiple genes.

A method for diagnosing breast cancer based on the detection of ctDNA methylation by MethyLight PCR, including the following steps:

Step 1. Use a cfDNA extraction kit to extract the cell-free DNA of the biological sample to be tested;

Step 2, carrying out bisulfite conversion to the DNA;

Step 3, the amplification primer of the above-mentioned DNA methylation molecular marker performs multiple PCR amplification on the transformed DNA to obtain a multiple PCR amplification product;

Step 4. The obtained multiplex PCR product is detected by multiplex fluorescence quantitative PCR with the probe for the above-mentioned DNA methylation molecular marker;

Step 5. Determine whether the sample is valid by the _CT value of the internal reference gene, and then use the _CT value of the internal reference gene to correct the _CT value of each molecular marker detected in the valid sample;

Step 6: Perform model analysis on the corrected data, and finally judge the benign and malignant pulmonary nodules.

In the step 5, if the _CT _value of the internal reference gene is between 8 and 18, the sample is judged as a valid sample; otherwise, it is an invalid sample; The _{CT value of the target DNA methylation molecular marker is corrected; if the CT} _value of the target DNA methylation molecular marker is less than 35, it is judged that the DNA methylation molecular marker has been detected, and the target DNA methylation molecular marker is obtained. Relative cycle number ΔC _T : ΔC _T = target DNA methylation molecular marker _CT value - internal reference gene _CT value; if the target DNA methylation molecular marker _CT value ≥ 35, the DNA methylation molecular marker is judged If the substance was not detected, it was assigned ΔC _T =27.

In the step 6, data analysis is performed according to the corrected ΔC _T value, and a breast cancer diagnosis model is established by using the Naive Bayes algorithm. Data analysis was carried out according to the corrected ΔC _T value, and the data set was randomly divided into training set and test set according to 6:4, and then the Naive Bayes algorithm was used for the combination of different DNA methylation molecular markers in the training set. The malignant prediction model was established, and finally the classification ability of the model was evaluated in the corresponding test set containing a combination of specific DNA methylation molecular markers.

The following specific examples further illustrate the present invention in detail, but are not intended to limit the protection scope of the present invention.

Example 1 This example provides a kit for detecting breast cancer in plasma samples, which contains 23 detection genes TLR5, C1orf61, NR2F1-AS1, ZDHHC1, OTP, FEZF2, TSHZ3, FLJ45513, POU3F1, NKX2- DNA methylation molecular marker Marker1 of 23 methylated regions in 1-AS1, PNPLA1, TIFAB, RADIL, EFCAB10, MAST1, ZFHX4-AS1, HOXB13, LHX5-AS1, LECT1, ALX3, BMP7, PRDM13 and ULBP1 Detection primers and probes to Marker23.

The kit designed three pairs of primers and three probes for each of the specific methylation sites in the 23 molecular markers Marker1 to Marker23 used for the detection of breast cancer in plasma samples (the probes can be fluorescently labeled with FAM, VIC, etc.). and NED and other fluorophore labels), and labeled as

combination

1, 2, 3, respectively. Wherein, the selected primer and probe combination in each molecular marker can be arbitrarily selected and combined with the

combination

1, 2 and 3 of primers and probes in other methylation markers and detected on the same platform. The specific primer and probe sequences corresponding to each molecular marker are shown in Table 1:

Table 1.1. Primer and probe sequences for related molecular markers

In this embodiment and the following embodiments, the primer and probe combination used is preferably the primer and probe combination 1 of Marker1-Marker23. In practical applications, the corresponding primers and probes will be selected according to different combinations of methylated molecular markers.

Example 2

This example uses the kit described in Example 1 to detect the methylation levels of 23 biomarkers in plasma samples.

A method for detecting methylation levels of DNA methylation molecular markers, comprising the following steps:

1. Extraction of cfDNA from the sample:

The specific operation steps of plasma DNA extraction were carried out according to the operation instructions of Life Company's MagMAX TM Cell-Free DNA Isolation Kit.

2. Sulfite conversion of the extracted cfDNA

The extracted cfDNA (10ng) was subjected to bisulfite conversion to deaminate the unmethylated cytosine into uracil, while the methylated cytosine remained unchanged to obtain a bisulfite converted The specific operation of transformation was carried out according to the instructions of Zymo Research's EZ DNA Methylation-Lightning Kit. Deamination of unmethylated cytosines in DNA to uracils, while methylated cytosines remain unchanged. All bisulfite-converted DNA products were used for multiplex PCR amplification.

3. Multiplex PCR amplification of transformed DNA

All the converted DNA products are amplified by multiplex PCR, and the reaction components are: a specific combination of molecular markers and a primer mixture of an internal reference gene, wherein the concentration of each primer is 200nM-300nM, and this embodiment is preferably 300nM The concentration of magnesium ions is 1-3mM, preferably 1.5mM in this embodiment; the concentration of dNTP mixture is 200-600uM, and this embodiment is preferably 400uM; the reaction enzyme is Phusion U (Thermo Fisher, Cat#F555L), a reaction The number of units is 1-3U, preferably 1.5U in this embodiment. The preparation of the multiplex PCR reaction system is shown in Table 2.1:

Table 2.1. Multiplex PCR reaction system

组分component	体积(ul)Volume (ul)	终浓度Final concentration
5X GC buffer 5X GC buffer	1010	1X1X
dNTP mix(10mM/dNTP)dNTP mix(10mM/dNTP)	22	400uM/dNTP400uM/dNTP
引物混合液(75uM/引物)Primer Mix (75uM/primer)	8.48.4	300nM/引物300nM/primer
MgCl2(25mM)MgCl2 (25mM)	33	1.5mM1.5mM
Phusion U(2U/ul)Phusion U(2U/ul)	0.750.75	1.5U1.5U

DNA模板DNA template	18-2218-22
DEPC H2ODEPC H2O	up to 50up to 50

The specific reaction conditions are: pre-denaturation, 98°C, 30s; 15-35 cycles of reaction, preferably 22 cycles in this embodiment: denaturation, 98°C, 15s; annealing, 58-66°C, 15-30s, this embodiment preferably 63°C, 15s; extension 72°C, 15-30s, preferably 15s in this embodiment.

4. Multiplex fluorescence quantitative PCR assay for multiplex PCR amplification products

The multiplex PCR products are diluted 1-5 times, preferably 3 times in this embodiment. The multiplex fluorescence quantitative PCR reaction components are: primer-probe mixture, wherein the concentration of each primer is 200-900nM, preferably 900nM in this embodiment; the probe concentration is 100-200nM, preferably 200nM in this embodiment. The reaction enzyme mixture used is 1 times the ChamQ Geno-SNP probe Master Mix (Vazyme, Cat#Q811-02), and one reaction is a 10ul system.

The primer-probe mixture in this system contains 2-3 molecular marker primers and corresponding probes labeled with different fluorophores. Table 2.2 lists the mixture of two molecular marker primers and probes. Mixed solution:

Table 2.2. The primer-probe mixture scheme in the multiplex fluorescence quantitative PCR reaction system

The specific reaction conditions are: pre-denaturation, 95°C, 5min; 40-50 cycles, preferably 45 cycles in this embodiment: denaturation, 95°C for 15s; annealing, 60-64°C, preferably 62°C, 1min in this embodiment, signal collect. The configuration of the qPCR fluorescence quantitative reaction system is shown in Table 2.3:

Table 2.3. qPCR fluorescence quantitative reaction system

_CT values of 23 molecular markers determined by multiplex fluorescence quantitative PCR using the fully methylated (positive control) and unmethylated (negative control) standards according to the mixed solution combination (mixed solution AM) shown in Table 5 , compared with the _CT value of the single fluorescence quantitative PCR reaction, the negative control was not detected in all combinations and single quantification. The _CT value of the fluorescence quantitative PCR reaction is similar to the _CT value of the fluorescence quantitative PCR reaction alone, and there is no significant difference. Therefore, it can be judged that the amplification efficiency of each molecular marker in the primer-probe mixture combination scheme of multiplex fluorescence quantitative PCR is not mutually exclusive. Interference, the quantitative performance is equivalent to that of a single area, and the simultaneous quantitative detection of 2-3 molecular markers can be achieved (Table 2.4).

Table 2.4. _CT values of each molecular marker under multiplex and single fluorescence quantitative PCR reactions

The present embodiment further provides a method for detecting breast cancer, further comprising the steps of:

5. Determine whether the test sample is a valid sample according to the _CT value of the internal reference gene in the sample determined by the fluorescence quantitative PCR reaction. If the _CT value of the internal reference gene in the test sample is between 8 and 18, the sample is judged as a valid sample. ; If the _CT value of the internal reference gene in the test sample is less than 8, the initial input amount of the sample is excessive; if the _CT value of the internal reference gene in the test sample is > 18, the initial sample input amount is insufficient. Samples with excess or insufficient initial input are judged as invalid samples and will not be included in testing and analysis.

6. On the premise that the sample is judged as a valid sample, if the C _T value of the target DNA methylation molecular marker is less than 35, it is judged that the DNA methylation molecular marker has been detected, and the target DNA methylation molecular marker is obtained. Relative cycle number ΔC _T : ΔC _T = target DNA methylation molecular marker _CT value - internal reference gene _CT value; if the target DNA methylation molecular marker _CT value ≥ 35, the DNA methylation molecular marker is judged If the substance was not detected, it was assigned ΔC _T =27.

7. Perform data analysis according to the corrected ΔCT of the target molecular markers in each sample, divide the data set into training set and test set according to 6:4, and perform 100 random segmentations. The combination of molecular markers uses the Naive Bayes algorithm to establish a benign and malignant prediction model, and finally evaluates the classification ability of the model in the corresponding test set containing a specific combination of molecular markers.

Example 3

This embodiment provides the detection of molecular markers in standard products, and the detection steps are as follows:

1. Standard preparation

1) 0% methylation standard preparation: use

Single Cell Kit (Qiagen, Cat#150343) and Mung Bean Nuclease (NEB, Cat#M0250L) treated NA12878 DNA to prepare 0% methylation standard;

2) 100% methylation standard preparation:

The prepared 0% methylation standard was treated with CpG Methyltransferase (M.SssI) to give a 100% methylation standard.

2. Preparation of standards with different methylation ratios:

Mix the 0% and 100% methylation standards in a gradient of the desired methylation ratio to obtain 0.2%, 0.4%, and 1% methylation ratio standards.

3. Perform bisulfite transformation on standard DNAs with different methylation ratios: the steps are as in Example 2, and the transformation input amount is 10-50 ng, preferably 50 ng in this embodiment.

4. Amplify the transformed standard DNA by multiplex PCR, as in Example 2. The multiplex PCR primer mixture is primers for 23 molecular markers and an internal reference gene.

5. Perform fluorescence quantitative PCR assay on the above-mentioned multiplex PCR amplification products, the steps are as in Example 2.

6. Determine whether the test sample is a valid sample according to the _CT value of the internal reference gene ACTB in the sample determined by the fluorescence quantitative PCR reaction. If the _CT value of the internal reference gene in the test sample is between 8 and 18, the sample is judged to be valid. sample;

7. On the premise that the sample is judged as a valid sample, if the C _T value of the target DNA methylation molecular marker is less than 35, it is judged that the DNA methylation molecular marker has been detected. If the target DNA methylation molecular marker C _T If the value is ≥35, it is judged that the DNA methylation molecular marker has not been detected.

In this example and the following examples, the primer-probe combination of each molecular marker is the preferred combination 1 in Example 1.

In this example and the following examples, a negative control is set for each test. The negative control uses water as a template to perform multiplex PCR, and the obtained negative control multiplex PCR products are then subjected to fluorescence quantitative PCR for each specific molecular marker. If there is no detection signal in the negative control, it is determined that there is no exogenous contamination in the entire experimental operation.

In this example, three completely independent repeated experiments were carried out, and 23 molecular markers all had detection signals in the 100% methylated standard, while there was no detection signal in the negative control and non-methylated detection standard. Take the amplification curve of a molecular marker (Marker1) as an example, as shown in Figure 1, among the standards with methylation ratio ≥ 0.2%, Marker1, Marker2, Marker3, Marker4, Marker8, Marker10, Marker11, Marker15, Marker17 , Marker19 and Marker21 all three tests have detection signals, indicating that the primers and probes of these molecular markers have a detection rate of 100% for samples with a methylation ratio of ≥0.2%; in each standard with a methylation ratio of ≥0.4% Among the samples, Marker5, Marker6, Marker7, Marker9, Marker12, Marker13, Marker14, Marker15, Marker18, Marker20, Marker22 and Marker23 all have detection signals, indicating that these probes and primers have a methylation ratio of ≥0.4%. The detection rate reached 100%; 22 molecular markers in the standard with a methylation ratio of 1% had detection signals in all three tests, indicating that the probes and primers of these molecular markers can detect methylation The ratio is 1% of the signal, the sensitivity is higher. Figure 4 is the amplification curve of the molecular marker (cg08599259) fluorescence quantitative PCR reaction detection.

Example 4 Performance of different molecular marker combinations for breast cancer detection in plasma samples

This example uses 23 molecular markers including Marker1 to Marker23 to detect any combination of primers and probes. Through the experimental method of Example 2, the specific detection kit, test method and data judgment processing are as described in Example 2. The primers The combination with the probe is the preferred combination in Example 1.

In this example, the molecular markers Marker1-Marker23 of 200 plasma samples were detected. The 200 plasma samples included 100 healthy controls and 100 breast cancer patients. The logistic regression model was used to detect the markers. For modeling analysis, the data set was randomly divided into training set and test set according to 6:4, and the benign and malignant prediction model was established by the naive Bayes algorithm.

The molecular marker combination A (Marker 1+internal reference) in Table 2.2 was selected for modeling analysis, and its AUC in the test set was 0.885 (specificity: 92.5%; sensitivity: 67.5%), and the ROC was shown in Figure 5a. It can be seen that the sensitivity and specificity of the PCR detection results of the primers and probes of Marker 1 are very good. Since 73% of malignant samples in the samples are early breast cancer samples, the combination of primers and probes to detect marker combination A also has high sensitivity and specificity for early breast cancer detection.

The molecular marker combination B in Table 2.2 was selected for modeling analysis, and its AUC in the test set was 0.8316 (specificity: 92.5%; sensitivity: 60%). The ROC is shown in Figure 5b, and the selected molecular markers can be seen The PCR detection results of the primers and probes also have good sensitivity and specificity. Since 73% of malignant samples are early breast cancer samples, the molecular marker combination also has high sensitivity and specificity for early breast cancer detection.

The molecular marker combination K in Table 2.2 was selected for modeling analysis, and its AUC in the test set was 0.7766 (specificity: 95%; sensitivity: 32.5%), and the ROC was shown in Figure 5c. It can be seen that the PCR detection results of the primers and probes of the selected molecular markers have better sensitivity and specificity, but the sensitivity is slightly lower than that of the D combination.

Molecular marker combinations A and B in Table 2.2 were selected for modeling analysis, and their AUC in the test set was 0.9144 (specificity: 92.5%; sensitivity: 72.5%), and the ROC was shown in Figure 6a. It can be seen that the detection result of the primer and probe combination of the selected molecular marker has better sensitivity and slightly weaker specificity than other combinations. Since 73% of malignant samples are early breast cancer samples, the molecular marker combination also has high sensitivity and specificity for early breast cancer detection.

The molecular marker combination A and random C and J in Table 2.2 were selected for modeling analysis, and the AUC in the test set was 0.8969 (specificity: 95%; sensitivity: 65%), and the ROC was shown in Figure 6b. It can be seen that the detection results of the primers and probes of the selected molecular markers have better sensitivity and slightly weaker specificity than other combinations. Since 73% of malignant samples in the samples are early breast cancer samples, the PCR detection results of the primers and probes of this molecular marker also have high sensitivity and specificity for early breast cancer detection.

The molecular marker combinations A and B in Table 2.2 were selected for modeling analysis and random C and J were used for modeling analysis. The AUC in the test set was 0.9163 (specificity: 95%; sensitivity: 75%), and the ROC was shown in Figure 6c . It can be seen that the PCR detection results of the primers and probes of the selected molecular markers have better sensitivity, and the specificity is slightly weaker than other combinations. Since 73% of the malignant samples are early breast cancer samples, the PCR detection results of the primers and probes of this molecular marker also have high sensitivity and specificity for early breast cancer detection.

All 23 molecular marker combinations were selected for modeling analysis, and their AUC in the test set was 0.9481 (specificity: 95%; sensitivity: 85%), and the ROC was shown in Figure 6d. It can be seen that the PCR detection results of the primers and probes of the selected molecular markers have better sensitivity and weaker specificity than other combinations. Since 73% of the malignant samples are early breast cancer samples, the PCR detection results of the primers and probes of this molecular marker also have high sensitivity and specificity for early breast cancer detection.

The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be regarded as the scope described in this specification.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

A detection kit for breast cancer-related methylation biomarkers, comprising: primers and probes selected from any of the following groups for the biomarker cg23035715: SEQ ID NO.1-SEQ ID NO.3, SEQ ID NO.70-SEQ ID NO.72, SEQ ID NO.139-SEQ ID NO.141.
The detection kit for breast cancer-related methylation biomarkers according to claim 1, further comprising primers and probes selected from at least one biomarker for cg26371731, cg04541368 and cg13973436; the The primers and probes for the cg26371731 biomarker are selected from: SEQ ID NO.4-SEQ ID NO.6, SEQ ID NO.73-SEQ ID NO.75, SEQ ID NO.142-SEQ ID NO.144;

The primers and probes for the cg04541368 biomarker are selected from: SEQ ID NO.7-SEQ ID NO.9, SEQ ID NO.76-SEQ ID NO.78, SEQ ID NO.145-SEQ ID NO.147 ;

The primers and probes for the cg13973436 biomarker are selected from: SEQ ID NO.10-SEQ ID NO.12, SEQ ID NO.79-SEQ ID NO.81, SEQ ID NO.148-SEQ ID NO.150 .
The detection kit for breast cancer-related methylation biomarkers according to claim 2, further comprising primers and probes selected from cg26371731, cg04541368 and cg13973436 biomarkers; the cg26371731 biomarkers The primers and probes of the marker are: SEQ ID NO.4-SEQ ID NO.6;

The primers and probes for the cg04541368 biomarker are: SEQ ID NO.7-SEQ ID NO.9;

The primers and probes for the cg13973436 biomarker are: SEQ ID NO.10-SEQ ID NO.12.
The detection kit for breast cancer-related methylation biomarkers according to claim 1 or 2, characterized in that it further comprises a kit selected from the group consisting of cg16304215, cg20072171, cg21501525, cg22778178, cg08599259, cg25566568, cg15634980, cg07458308, cg01348584, Primers and probes for at least one biomarker of cg14140881, cg25756435, cg00594560, cg18087672, cg14868703, cg17632299, cg18786873, cg20631750, cg25924096, and cg15321298;

The primers and probes for the cg16304215 biomarker are selected from: SEQ ID NO.13-SEQ ID NO.15, SEQ ID NO.82-SEQ ID NO.84, SEQ ID NO.151-SEQ ID NO.153 ;

The primers and probes for the cg20072171 biomarker are selected from: SEQ ID NO.16-SEQ ID NO.18, SEQ ID NO.85-SEQ ID NO.87, SEQ ID NO.154-SEQ ID NO.156 ;

The primers and probes for the cg21501525 biomarker are selected from: SEQ ID NO.19-SEQ ID NO.21, SEQ ID NO.88-SEQ ID NO.90, SEQ ID NO.157-SEQ ID NO.159 ;

The primers and probes for the cg22778178 biomarker are selected from: SEQ ID NO.22-SEQ ID NO.24, SEQ ID NO.91-SEQ ID NO.93, SEQ ID NO.160-SEQ ID NO.162 ;

The primers and probes for the cg08599259 biomarker are selected from: SEQ ID NO.25-SEQ ID NO.27, SEQ ID NO.94-SEQ ID NO.96, SEQ ID NO.163-SEQ ID NO.165 ;

The primers and probes for the cg25566568 biomarker are selected from: SEQ ID NO.28-SEQ ID NO.30, SEQ ID NO.97-SEQ ID NO.99, SEQ ID NO.166-SEQ ID NO.168 ;

The primers and probes for the cg15634980 biomarker are selected from: SEQ ID NO.31-SEQ ID NO.33, SEQ ID NO.100-SEQ ID NO.102, SEQ ID NO.169-SEQ ID NO.171 ;

The primers and probes for the cg07458308 biomarker are selected from: SEQ ID NO.34-SEQ ID NO.36, SEQ ID NO.103-SEQ ID NO.105, SEQ ID NO.172-SEQ ID NO.174 ;

The primers and probes for the cg01348584 biomarker are selected from: SEQ ID NO.37-SEQ ID NO.39, SEQ ID NO.106-SEQ ID NO.108, SEQ ID NO.175-SEQ ID NO.177 ;

The primers and probes for the cg14140881 biomarker are selected from: SEQ ID NO.40-SEQ ID NO.42, SEQ ID NO.109-SEQ ID NO.111, SEQ ID NO.178-SEQ ID NO.180 ;

The primers and probes for the cg25756435 biomarker are selected from: SEQ ID NO.43-SEQ ID NO.45, SEQ ID NO.112-SEQ ID NO.114, SEQ ID NO.181-SEQ ID NO.183 ;

The primers and probes for the cg00594560 biomarker are selected from: SEQ ID NO.46-SEQ ID NO.48, SEQ ID NO.115-SEQ ID NO.117, SEQ ID NO.184-SEQ ID NO.186 ;

The primers and probes for the cg18087672 biomarker are selected from: SEQ ID NO.49-SEQ ID NO.51, SEQ ID NO.118-SEQ ID NO.120, SEQ ID NO.187-SEQ ID NO.189 ;

The primers and probes for the cg14868703 biomarker are selected from: SEQ ID NO.52-SEQ ID NO.54, SEQ ID NO.121-SEQ ID NO.123, SEQ ID NO.190-SEQ ID NO.192 ;

The primers and probes for the cg17632299 biomarker are selected from: SEQ ID NO.55-SEQ ID NO.57, SEQ ID NO.124-SEQ ID NO.126, SEQ ID NO.193-SEQ ID NO.195 ;

The primers and probes for the cg18786873 biomarker are selected from: SEQ ID NO.58-SEQ ID NO.60, SEQ ID NO.127-SEQ ID NO.129, SEQ ID NO.196-SEQ ID NO.198 ;

The primers and probes for the cg20631750 biomarker are selected from: SEQ ID NO.61-SEQ ID NO.63, SEQ ID NO.130-SEQ ID NO.132, SEQ ID NO.199-SEQ ID NO.201 ;

The primers and probes for the cg25924096 biomarker are selected from: SEQ ID NO.64-SEQ ID NO.66, SEQ ID NO.133-SEQ ID NO.135, SEQ ID NO.202-SEQ ID NO.204 ;

The primers and probes for the cg15321298 biomarker are selected from: SEQ ID NO.67-SEQ ID NO.69, SEQ ID NO.136-SEQ ID NO.138, SEQ ID NO.205-SEQ ID NO.207 .
The detection kit for breast cancer-related methylation biomarkers according to claim 1 or 2, further comprising primers and probes for an internal reference gene, wherein the primers and probes for the internal reference gene are : SEQ ID NO.208-SEQ ID NO.210.
The detection kit for breast cancer-related methylation biomarkers according to claim 4, further comprising: Primers and probes for cg25756435, cg00594560, cg18087672, cg14868703, cg17632299, cg18786873, cg20631750, cg25924096, and cg15321298.
The detection kit for breast cancer-related methylation biomarkers according to claim 6, wherein,

The primers and probes for the cg16304215 biomarker are SEQ ID NO.13-SEQ ID NO.15;

The primers and probes for the cg20072171 biomarker are SEQ ID NO.16-SEQ ID NO.18;

The primers and probes for the cg21501525 biomarker are SEQ ID NO.19-SEQ ID NO.21;

The primers and probes for the cg22778178 biomarker are SEQ ID NO.22-SEQ ID NO.24;

The primers and probes for the cg08599259 biomarker are SEQ ID NO.25-SEQ ID NO.27;

The primers and probes for the cg25566568 biomarker are SEQ ID NO.28-SEQ ID NO.30;

The primers and probes for the cg15634980 biomarker are SEQ ID NO.31-SEQ ID NO.33;

The primers and probes for the cg07458308 biomarker are SEQ ID NO.34-SEQ ID NO.36;

The primers and probes for the cg01348584 biomarker are SEQ ID NO.37-SEQ ID NO.39;

The primers and probes for the cg14140881 biomarker are SEQ ID NO.40-SEQ ID NO.42;

The primers and probes for the cg25756435 biomarker are SEQ ID NO.43-SEQ ID NO.45;

The primers and probes for the cg00594560 biomarker are SEQ ID NO.46-SEQ ID NO.48;

The primers and probes for the cg18087672 biomarker are SEQ ID NO.49-SEQ ID NO.51;

The primers and probes for the cg14868703 biomarker are SEQ ID NO.52-SEQ ID NO.54;

The primers and probes for the cg17632299 biomarker are SEQ ID NO.55-SEQ ID NO.57;

The primers and probes for the cg18786873 biomarker are SEQ ID NO.58-SEQ ID NO.60;

The primers and probes for the cg20631750 biomarker are SEQ ID NO.61-SEQ ID NO.63;

The primers and probes for the cg25924096 biomarker are SEQ ID NO.64-SEQ ID NO.66;

The primers and probes for the cg15321298 biomarker are SEQ ID NO.67-SEQ ID NO.69.
The detection kit for breast cancer-related methylation biomarkers according to claim 1 or 2, further comprising a PCR reaction solution comprising DNATaq polymerase, dNTPs, Mg and 10× DNA polymerase buffer.
The detection kit for breast cancer-related methylation biomarkers according to claim 1 or 2, wherein the sample to be detected by the detection kit is blood, serum or plasma, or a tissue sample.
The detection kit for breast cancer-related methylation biomarkers according to claim 1 or 2, further comprising a primer mixture, wherein in the amplification system, the concentration of each primer is 300±10nM; The concentration of magnesium ions is 1.5±0.1mM, the concentration of dNTP mixture is 400±10uM; the reaction enzyme is Phusion U; the primer-probe mixture, in the multiplex fluorescence quantitative PCR reaction: the concentration of each primer is 900±10nM; the probe Concentrations were at 200 ± 10 nM.