A breast cancer gene methylation marker and its application

Abstract

The present invention discloses a breast cancer gene methylation marker and its application, and belongs to the technical field of disease diagnosis and prevention. The present invention screens breast cancer by detecting the methylation degree of free circulating tumor DNA (ctDNA) tumor suppressor genes in peripheral blood plasma or serum by an automated high-throughput instrument, specifically comprising: screening breast cancer differential methylation genes in a database, using the MethyLight method, and using digital PCR to detect breast cancer-specific methylation genes. The method of the present invention has the characteristics of high sensitivity, good specificity, non-invasive sampling, repeated monitoring, and automated detection. The method of the present invention is suitable for large-scale screening of breast cancer, and provides a new technical means for screening of breast cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of disease diagnosis and prevention, and specifically relates to a breast cancer gene methylation marker and application thereof. Background Art

[0002] Breast cancer has become the most common malignant tumor that seriously threatens women's health. According to data from the World Health Organization's International Agency for Research on Cancer (IARC), the number of new cancer cases in the world in 2020 totaled 19.29 million, of which 2.26 million were new breast cancer patients. A large amount of evidence-based medicine shows that breast cancer screening can increase the early diagnosis rate of breast cancer and reduce the mortality rate.

[0003] At present, clinical breast imaging examinations are relatively common in my country. For suspicious results found in screening, further specialist diagnosis or follow-up is required, and histopathological examination is required to confirm or exclude the diagnosis of breast cancer.

[0004] Breast ultrasound is an ultrasonic imaging technique for breast tissue that has good resolution for distinguishing cystic and solid breast lesions. Compared with breast X-ray examination (mammography), breast ultrasound is a non-invasive test method without radiation and does not cause pain or discomfort. When guiding puncture biopsy or other therapeutic interventions, B-ultrasound can provide real-time images to help doctors locate lesions more accurately. Breast ultrasound has its own unique disadvantages. For women with higher breast density, B-ultrasound accuracy may be poor; B-ultrasound is affected by operator experience, requires instruments and equipment, and takes a long time to operate, which is not suitable for large-scale screening; B-ultrasound is not sensitive enough for small, deep lesions. Mammography carries the risk of radiation exposure. Especially in the case of long-term screening, cumulative radiation exposure is a risk. Due to the presence of radiation, mammography is generally not recommended for pregnant women because radiation may have adverse effects on the fetus. The resolution of X-ray photography is relatively limited, which may make some small or early-stage lesions difficult to detect. Especially for women with denser breast tissue, the sensitivity of X-ray photography may be low. Dense breast tissue may appear white on X-ray images, making the lesions difficult to observe. Breast MR imaging (MRI) performs three-dimensional scanning of the breast in its natural state to evaluate the morphological characteristics of the lesions in a three-dimensional manner. It can be used as an important supplementary examination method in addition to breast ultrasound and X-ray examinations. However, breast MR equipment is expensive, the examination costs are high, and the training period for imaging physicians is long, which makes it difficult to promote grassroots screening.

[0005] DNA methylation occurs by modifying DNA by adding a methyl group to the 5th carbon atom of cytosine. CpG is usually present at a high density in the genome, forming CpG islands. Methylation of CpG islands in gene promoter regions regulates gene expression, because high methylation CpG islands in genes can lead to inhibition of gene transcription - especially by downregulating tumor suppressor genes - and lead to the occurrence and development of cancer. Abnormal DNA methylation in tumors serves as an early diagnostic and prognostic marker. Studies on changes in DNA methylation in plasma and serum have shown that DNA methylation at gene-specific sites may provide a promising alternative for non-invasive breast cancer screening.

[0006] Digital PCR (ddPCR) is a technology used in the field of biology to amplify and detect DNA. Compared with traditional quantitative PCR, digital PCR can handle a wider range of target concentrations because it is not limited by PCR amplification efficiency. This is very beneficial for low-abundance methylation detection in plasma and serum. In addition, digital PCR can provide more accurate quantitative analysis because it can directly count the number of DNA molecules in each PCR reaction, making experimental design and data analysis simpler and the results more accurate. Since digital PCR does not rely on estimates of PCR efficiency, it is less affected by inhibitory substances (such as compounds that inhibit PCR that may be present in the sample), which makes digital PCR more robust.

[0007] In view of this, it is of great significance to study a new method of using digital PCR to detect breast cancer-specific methylation genes for breast cancer screening. Summary of the invention

[0008] In view of the defects and deficiencies in the existing breast cancer screening technology, the present invention provides a breast cancer gene methylation marker and its application. The present invention detects the methylation degree of free circulating tumor DNA (ctDNA) tumor suppressor genes in peripheral blood plasma or serum by an automated high-throughput instrument to screen for breast cancer, specifically including: screening breast cancer differential methylation genes in the database, using the MethyLight method, and using digital PCR to detect breast cancer-specific methylation genes. The method of the present invention has the characteristics of high sensitivity, good specificity, non-invasive sampling, repeated monitoring, and automated detection. The method of the present invention is suitable for large-scale screening of breast cancer and provides a new technical means for screening of breast cancer.

[0009] The purpose of the present invention is mainly reflected in the following three aspects:

[0010] In a first aspect, the present invention provides a primer and / or probe for specific methylation detection of breast cancer.

[0011] In a second aspect, the present invention provides a specific methylation detection method for diagnosing breast cancer, the method comprising the specific primers and / or probes described above.

[0012] In a third aspect, the present invention also provides the use of the above-mentioned specific primers and / or probes in the preparation of detection of breast cancer.

[0013] In order to achieve the above object, the technical means adopted by the present invention are:

[0014] In a first aspect, the present invention provides a primer and / or probe for specific methylation detection of breast cancer.

[0015] The present invention provides a gene methylation marker for early screening and diagnosis of endometrial cancer, which is selected from the regions chr20:2781212-2781649, chr6:10881846-10882051, chr11:31848487-31848976, chr10:22634000-22634862, chr1:67218079-67218293, chr5:150284385-150284635 of target genes CPXM1, GCM2, PAUPAR, SPAG6, TCTEX1D1, and ZNF300, and a primer probe for detecting gene methylation is designed.

[0016] Furthermore, the target gene and the corresponding chromosome region are shown in the following table:

[0017]

[0018]

[0019] Furthermore, the present invention designs primer probes for MethyLight PCR amplification in Beacon Designer 8 software for the above-mentioned target genes and regions.

[0020] Furthermore, the sequences of the primer probes are shown in the following table:

[0021] Detect the base sequences of the front and back primers and probes of each methylated target gene region

[0022]

[0023]

[0024]

[0025]

[0026] Furthermore, the melting temperature (Tm) of the primer probe is shown in the following table:

[0027]

[0028]

[0029] Furthermore, since methylated (M) and unmethylated (UM), methylated (M) and internal reference gene (IC) will react in the same PCR tube, different fluorescent groups and quenching groups can be designed.

[0030] Furthermore, the (LNA) in the base (A, T, C or G) indicates a locked nucleic acid base, which can increase the Tm value, increase the binding affinity with the DNA single strand, and have higher sequence specificity.

[0031] Furthermore, the Tm value is automatically calculated by the software Beacon Designer 8. The difference between the Tm values ​​of the front and rear primers is small, and the Tm value of the probe is about 10°C higher than that of the primer, ensuring the PCR amplification efficiency. At the same time, the Tm value of the primer provides a reference for the annealing temperature setting during PCR amplification.

[0032] Furthermore, the PCR reaction procedure of the CPXM1 gene region is as follows:

[0033]

[0034] Furthermore, the methylation region sequence of the gene CPXM1 is selected from the DNA strand in UCSC Genome Browser on Human (GRCh37 / hg19), hg19_dna range=chr20:2781212-27816495'pad=03'pad=0strand=+; the sequence of the DNA strand is as follows:

[0035] CCCACATGGCGGGGATTGAGTGCCAGGGCGCGGGCTACGGCGGGTGGCGGGTCGGTCTCTTCCTGCCGAGTGCGCCGAGCCCCCCGCCCCTTCCTGCCCCCCGCCCCTCCGCAGCCTCCTGCCCGCCCACAGCCCGCTGCCCGCCTCGGACCTCGGAGAGGAGGAGAGGGCCGGGAGGAGCGAGCGGGGCTGACCTCCAAGGGGCCGCCCCAAGTC TCAGGACCGCCGACTCTGCCCTTCCTCTCCGCCGTCTGGGGCCAACCCGCCTCGTTGGCGCGCTGCGCCTTTATAGTCTGCAAAGCCACACTGAAGAGACCAGAGATCACCTGGTGGCCATCAAGTCGACCAACAGGAGGCAAAGCCCCTTATCTGAGCAATTCAGAATGACACTTCCCTATTGTCTAAAGCCACATCTGGTACCAAGCTTCCGTCCCA.

[0036] Furthermore, all C in the CG in the sequence of the DNA chain in the methylated region sequence of the gene CPXM1 are methylated and subjected to bisulfite conversion, wherein the unmethylated C is converted to T and the methylated C remains unchanged, thereby obtaining a converted DNA chain. Furthermore, the sequence of the converted DNA chain is as follows:

[0037] TTTATATGGCGGGGATTGAGTGTTAGGGGCGCGCGGGTTACGGCGGGTGGCGGGTCGGTTTTTTTTTGTCGAGTGCGTCGAGTTTTTCGTTTTTTTGTTTTTCGTTTTTTCGTAGTTTTTTGTTCGTTTA TAGTTCGTTGTT CGTTTCGGATTTCG GAGAGGAGGAGAGGGT CGGGAGGAGCGAGCGGGGTTGATTTT TAAGGGGT CGTTTTAAGTTT TAGGATCGTCGATTTTGTTTTTTTTTTTCGTCGTTTGGGGTTAATTCGTTTCGTTGGCGCGTTGCGTTTTTATAGTTTGTAAAGTTATATTGAAGAGATTAGAGATTATTTGGTGGTTATTAAGTCGATTAATAGGAGGTAAAGTTTTTTATTTGAGTAATTTAGAATGATATTTTTTATTGTTTAAAGTTATATTTGGTATTAAGTTTTCGTTTTA.

[0038] Furthermore, the underlined portion in the converted DNA chain sequence is the position of the methylation primer probe sequence of CPXM1 (chr20: 2781212-2781649) finally obtained.

[0039] Furthermore, in the sequence of the converted DNA chain in the methylated region sequence of the gene CPXM1, all C in CG in the converted DNA chain sequence is not methylated, and bisulfite conversion is performed, and the unmethylated C is converted to T, and the methylated C remains unchanged, thereby obtaining a converted DNA chain; further, the sequence of the converted DNA chain is as follows:

[0040] TTTATATGGTGGGGATTGAGTGTTAGGGGTGTGTGGGTTATGGTGGGTGGTGGGTTGGTTTTTTTGTTGAGTGTGTTGAGTTTTTTGTTTTTTTTTGTTTTGTTTTTTTGTAGTTTTTTGTTTGTTTATAGTTTG TTGTT TGTTTTGGATTTTGGAGAGGA GGAG AGGGTTGGGAGGAGTGAGTGGGGTTGATTT TTAAG GGGTTGTTTTAAGTTT TAGGATTGTTGATT TTGTTTTTTTTTGTTGTTTGGGGTTAATTTGTTTTGTTGGTGTGTTGTGTTTTTATAGTTTGTAAAGTTATATTGAAGAGATTAGAGATTATTTGGTGGTTATTAAGTTGATTAATAGGAGGTAAAGTTTTTTATTTGAGTAATTTAGAATGATATTTTTTATTGTTTAAAGTTATATTTGGTATTAAGTTTTTGTTTTA.

[0041] Furthermore, the underlined portion in the converted DNA chain sequence is the position of the unmethylated primer probe sequence of CPXM1 (chr20: 2781212-2781649) obtained in the end.

[0042] Furthermore, the PCR reaction procedure of the GCM2 gene region is as follows:

[0043]

[0044] Further, the methylation region sequence of the gene GCM2 is selected from the DNA strand in UCSC Genome Browser on Human (GRCh37 / hg19), hg19_dna range=chr6:10881646-108822515'pad=03'pad=0strand=+; further, the sequence of the DNA strand is as follows:

[0045] .

[0046] Furthermore, all C in CG in the DNA sequence in the methylated region sequence of the gene GCM2 is methylated, and bisulfite conversion is performed, unmethylated C is converted to T, and methylated C remains unchanged, and the sequence of the converted DNA chain is as follows:

[0047] TTTTTAAAGTGTTGGGATTATAGGTATGAATTATAGCGTTCGGTTAAGAAGATATATTTTAAATTTAATTTAGTTTTTTGTAGAGTTTAGTTTTTTTTTTATATTTGAGAGGGTTTTAGAAATTTAG AAATTTTGCGGGTATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGGTTCGTTCGTAGATTTTTTAAGAATTCGAATGTTATTTTTTTTTTTCGGATGGATAGC GTTTCGCGTGTTCGTATATATTCG GTTTATTT ATTTGAG GTATTTGCGGATCGTTGATGTTT T AGTTGAGTTGTATTTCGTAGGAGTATA CGTCGATCGTTTTTTGTATCGCGGTCGTCGGTATTTGTTTAATTCGTTCGCGTTTTTCGTTTAGGGTTTTGAAAAATAGAAGAAAAAAGGATAGGTGCGTTAGGTGGGTTTTTTTTTTTTTTTAAAGA AGAAAGTGGGGTGTGTGAAGGGGAGGTGTAGAGAGAGAAAGAGAGAGAGGTTGTTTAGATATTTGGAAGTTGGGGATAATGGTTATGGATTCGGGCGGGGTTTTGTTTTTGGTCGCGGTGTTGAACG.

[0048] Furthermore, the underlined portion in the converted DNA chain sequence is the position of the methylation primer probe sequence of GCM2 (chr6: 10881646-10882251) finally obtained.

[0049] Furthermore, all C in CG in the DNA sequence in the methylated region sequence of the gene GCM2 is not methylated, and bisulfite conversion is performed, and the unmethylated C is converted to T, and the methylated C remains unchanged. The converted DNA chain sequence is as follows: In the DNA sequence:

[0050] TTTTTAAAGTGTTGGGATTATAGGTATGAATTATAGTGTTTGGTTAAGA

[0051] AGATATATTTTTAAATTTAATTTAGTTTTTTGTAGAGTTTAGTTTTTTTTTT

[0052] ATATTTGAGAGGGTTTTAGAAATTTAGAAATTTTGTGGGTATGTGTGTGTG

[0053] TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGTTTG

[0054] TTTGTAGATTTTTTAAGAATTTGAATGTTATTTTTTTTTTTTTGGATGGATA

[0055] GT GTTTTGTGTGTTTGTATATATTTGGT TTATTT ATTTGAGGTATTTGTGGA

[0056] TTGTTGATGTTT TA GTTGAGTTGTATTTTGTAGGAGTATAT GTTGATTGTTT

[0057] TTTGTATTGTGGTTGTTGGTATTTGTTTAATTTGTTTGTGTTTTGTTTAG

[0058] GGTTTTGAAAAATAGAAGAAAAAAGGATAGGTGTGTTAGTGGGTTTTTT

[0059] TTTTTTTTTTAAAGAAGAAAGTGGGGTGTGTGAAGGGGAGGTGTAGAGA

[0060] GAGAGAAAGAGAGAGAGGTTGTTTAGATATTTGGAAGTTGGGGATAATGGTTATGGATTTGGGTGGGGTTTTGTTTTTGGTTGTGGTGTTGAATG.

[0061] Furthermore, the underlined portion in the converted DNA chain sequence is the position of the unmethylated primer probe sequence of GCM2 (chr6: 10881646-10882251) obtained in the end.

[0062] Furthermore, the PCR reaction procedure of the PAUPAR gene region is as follows:

[0063]

[0064] Furthermore, the methylation region sequence of the gene PAUPAR is selected from the DNA strand in UCSC Genome Browser on Human (GRCh37 / hg19), hg19_dna range=chr11:31848487-318489765'pad=03'pad=0strand=+; the sequence of the DNA strand is as follows:

[0065] ACGCAGGCGGCGGGAGTTGCACCGCCACAGCCCGGGAGACCAAAGAGTCTCCACTCCCCGCTCCCGGCGCCTCCTCGCCGACCGGAGCCCCGAGCCCAGTGAGCCCAGCCGGTCCCACGTCGAGAGGCTGGGTTGGGAGAAGTTCTGCTGGTCGTGGGCTCGGCCCCCAGGCGCCAGGCCGAGTGCCCACCTCGGCTTCTTTAGGGGGCCTACAGCGGGCAGCCGAGCGGCTGGCGGACCCGG AGCTTGGGAGGCGACCGCCAGGCTGGTGCCCGGCCTGACCCGGCGTTCGCGGCCGCCCGTCCGCCCGCCGCGGGCCGGGAGCGTACAGGAGTGTGACGCAGATTGTGAAAACAGAAGGGAGGGAGTTGGGTCATTTCCTTCGCTAATCATCGCCCCCTCCTGCTCCTCCTCCCCACCCCCATCCCATCCTCGCCGCCACCCAGCCTCGAACTCTCTTCCTTGCCAGAGTCTGCCTCACTTTGAGC.

[0066] Furthermore, all C in the CG in the DNA chain sequence in the methylated region sequence of the gene PAUPAR are methylated, and bisulfite conversion is performed, unmethylated C is converted to T, and methylated C remains unchanged, and the sequence of the converted DNA chain is as follows:

[0067] ACGTAGGCGGCGGGAGTTGTATCGTTATAGTTCGGGAGATTAAAGAGTTTTTATTTTTCGTTTTCGGCGTTTTTTCGTCGATCGGAGTTTCGAGTTTAGTGAGTTTAGTCGGTTTTACGTCGAGAGGCGTTGGGTTGGGAGAAGTTTTGTTGGTCGTGGGTTCGGTTTTTAGGCGTTAGGTCGAGTGTTTATTTCGGTTTTTTTTAG GGGGTTTATAGCGG GTAGTCG AGC GGTTGGCGGATTCGGAGTTTGGGAG GCGATCGTTA GGTTGGTGTTCGGTTTGATTCG GCGTTCGCGGTCGTTCGTTCGTTCGTCGCGGGTCGGGAGCGTATAGGAGTGTGACGTAGATTGTGAAAATAGAAGGGAGGGAGTTGGGTTATTTTTTTCGTTAATTATCGTTTTTTTTTGTTTTTTTTTTATTTTTTATTTTATTTCGTCGTTATTTAGTTTCGAATTTTTTTTTTTGTTAGAGTTTGTTTTATTTTGAGT.

[0068] Furthermore, the underlined portion in the converted DNA chain sequence is the position of the methylation primer probe sequence of the final PAUPAR (chr11: 31848487-31848976).

[0069] Furthermore, in the sequence of the converted DNA chain in the methylated region sequence of the gene PAUPAR, all C in CG in the converted DNA chain sequence is not methylated, and bisulfite conversion is performed, and the unmethylated C is converted to T, and the methylated C remains unchanged, thereby obtaining a converted DNA chain; further, the sequence of the converted DNA chain is as follows:

[0070] ATGTAGGTGGTGGGAGTTGTATTGTTATAGTTTGGGAGATTAAAGAGTTTTT

[0071] ATTTTTTGTTTTTGGTGTTTTTTTGTTGATTGGAGTTTTGAGTTTAGTGAGTT

[0072] TAGTTGGTTTTATGTTGAGAGGTGTTGGGTTGGGAGAAGTTTTGTTGGTTG

[0073] TGGGTTTGGTTTTTAGGTGTTAGGTTGAGTGTTTATTTTGGTTTTTTTAG GG

[0074] GGTTTATAGTGGGTAGTTGAGTGGTTGGTGGATTTGGAGTTTGGGAGGTGA

[0075] TTGT TAGGTTGGTGTTTGGTTTGATTTG GTGTTTGTGGTTGTTTGTTTGTTT

[0076] GTTGTGGGTTGGGAGTGTATAGGAGTGTGATGTAGATTGTGAAAATAGAAG

[0077] GGAGGGAGTTGGGTTATTTTTTTTGTTAATTATTGTTTTTTTTTGTTTTTTTTT

[0078] TTTATTTTTTATTTTATTTTTGTTGTTATTTAGTTTTGAATTTTTTTTTTTGTTAGAGTTTGTTTTATTTTGAGT.

[0079] Furthermore, the underlined portion in the converted DNA chain sequence is the position of the unmethylated primer probe sequence of PAUPAR (chr11: 31848487-31848976) obtained in the end.

[0080] Further, the PCR reaction procedure of the SPAG6 gene region is as follows:

[0081]

[0082]

[0083] Furthermore, the methylation region sequence of the gene SPAG6 is selected from the DNA strand in UCSC Genome Browser on Human (GRCh37 / hg19), hg19_dna range=chr10:22634000-226348625'pad=03'pad=0strand=+; the sequence of the DNA strand is as follows:

[0084] TCGGATTGTACAATGTGACGTGTGAGATTTCTCGGGACCGTCGAAGCTCAGACAACTCCAGGGCTGCAATTTACTCGTCTGGCTGCCCTCGAACCAGGCTCCCTTGGCGCGTGGGGCTCTCCCGGCGCTTGGTCCCGACCCGCGCCGCAGTCAGGATACCACGGCAATCTGGGGTCGCTTTGTAAGGAGCTCCGACCTCCGGGCAGTGCAGGGATACTCGACGGCGCGCGATGTCCCGGCGGCTCCGCGAGCCTGGCGCGCTGAGAGCCCCAGCAGTCCGAGTCCTCGAGCTCGGGCGTCTTCGCGCCGCCGCCCCGCTCAGTGCGCCCAGGCACCGCGGCCGTGACGTCACGCCCGGGACTGGCCGTTGCAGCAAGACGGCCGCGTTCCGGTTCCGGTAGGTTGCCCGGGAGACGCGGGTACACAGAGAAGCGGCTCCCGTCGGAGGCCGAGTCGTCGCCACGATCGCCCCCTTGGTGGACTCGCAGGCCGAGCGGCTTCCCCGCAGAGCTCGAGGAGGGCAGACGGCGGCGGGGGCGCCATGAGTCAGAGGCAGGTGCTGCAAGGTAGGGCCGAGGCGGGCAGGTGCCCTAACTAGCTGGCGCCGAGGAGACCCGGGTGCGGTGGGCTCCACCGACTCTCTCTCCCGCAGTGTTCGAGCAATACCAGAAGGCCAGGACCCAGTTCGTGCAGATGGTGGCGGAGCTGGCGACTAGACCCCAAAACATCGAGACGCTGCAGAACGCGGGTGAGCCCGGAGCCCGAACCCCCGTCGCCCCCCGCGCACTGAGTCCCCGACGCCTCCGCCCCGCTGCCCTGCCCGTGGAGCTCTTGGGGAGCCGCAGTGTGGGGACCGGAGTTCG.

[0085] Furthermore, all C in the CG in the sequence of the DNA chain in the methylated region sequence of the gene SPAG6 are methylated and subjected to bisulfite conversion, wherein the unmethylated C is converted to T and the methylated C remains unchanged, thereby obtaining a converted DNA chain. Furthermore, the sequence of the converted DNA chain is as follows:

[0086] TCGGATTGTATAATGTGACGTGTGAGATTTTTCGGGATCGTCGAAGTTTAGATAATTTTAGGGTTGTAATTTATTCGTTTTGGTTGTTTTCGAATTAGGTTTTTTTGGCGCGTGGGGTTTTTTCGGCGTTTGGTTTCGATTCGCGTCGTAGTTAGGAT ATTACGGTAATTTGGGGTCGTT TTGTAAGGAGTTTCGATTTT CGGGTAGTGTAGGGATATTCG ACGGCG CGCGAT GTTTCGGCGGTTTCGCG .

[0087] Furthermore, the underlined portion in the converted DNA chain sequence is the position of the methylation primer probe sequence of SPAG6 (chr10: 22634000-22634862) finally obtained.

[0088] Furthermore, in the sequence of the converted DNA chain in the methylated region sequence of the gene SPAG6, all C in CG in the converted DNA chain sequence is not methylated, and bisulfite conversion is performed, and the unmethylated C is converted to T, and the methylated C remains unchanged, thereby obtaining a converted DNA chain; further, the sequence of the converted DNA chain is as follows:

[0089] TTGGATTGTATAATGTGATGTGTGAGATTTTTTGGGATTGTTGAAGTTTAGATAATTTTAGGGTTGTAATTTATTTGTTTTGGTTGTTTTTGAATTAGGTTTTTTTGGTGTGTGGGGTTTTTTTGGTGTTTGGTTTTGATTTGTGTTGTAG TTAGGATATTATGGTAATTTGGGGTTG TTTTGTAAGGAGTTTTGATTTTTGGGT AGTGTAGGGATATTTG ATGGTGTGTGATG T TTTGGTGGTTTTGTGAGTTTGG.

[0090] Furthermore, the underlined portion in the above converted DNA chain sequence is the position of the unmethylated primer probe sequence of SPAG6 (chr10: 22634000-22634862) finally obtained.

[0091] Furthermore, the PCR reaction procedure of the TCTEX1D1 gene region is as follows:

[0092]

[0093]

[0094] Furthermore, the methylation region sequence of the gene TCTEX1D1 is selected from the DNA strand in UCSC Genome Browser on Human (GRCh37 / hg19), hg19_dna range=chr1:67217979-672183935'pad=03'pad=0strand=-; the sequence of the DNA strand is as follows:

[0095] GGAGAGGAGGAATAAGGTGGCGGAGAGATCGGGGGCCAAGTAGGGCCATTTCTCAACAAAGGGCCGATCCCTTGCCCCCTATCCTTCCTTTGGGGTCCAGACGCGCTGCAGAGAGAGGCGACCCTACCTGCAGCAGGGAGGCTTCGCCCGGAGCCTCCCAGACCTCCGGCACTGCAGACACTGCGCGCGGCGCGGCTCCCGCGTCCCAG GCTTCATTCAGCCGGCGGCCGCCCGCGCACTCTCCCTGGACTCTGAGGCCGGTCTCCAGGGTGACCTCAAACAGAGCCCTCGGCCCTCCCCGGCCGGACTTGGCCGCTGAGTGGCCCTCTGAGCTGCCCTAAGGATGTAGAGGATCTCATCTTCACTGCCCACTGGGGGTCACTCTGAGCCGGGCTGCGTCCTGGGTTGGCGGCGAG.

[0096] Furthermore, all C in the CG in the sequence of the DNA chain in the methylated region sequence of the gene TCTEX1D1 are methylated, and bisulfite conversion is performed, wherein the unmethylated C is converted to T, and the methylated C remains unchanged, to obtain a converted DNA chain. Furthermore, the sequence of the converted DNA chain is as follows:

[0097] GGAGAGGAGGAATAAGGTGGCGGAGAGATCGGGGTTAAGTAGGGTTATTTTTTAATAAAGGGTCGATTTTTTGTTTTTTATTTTTTTTTTGGGGTTTAGAC GCGTTGTAGAGAGAGGCGAT TTTATTT GTAGTAGGGAGGTTTC GTTCGGAGTTT TTTAGATTT TCGGTATTGTAGATATTGCGCGCGGCCGCGGTTTTCGCGTTTTAGGTTTTATTTAGTCGGCGGTCGTTCGCGTATTTTTTTTGGATTTTGAGGTCGGTTTTTAGGGTGATTTTAAATAGAGTTTTCGGTTTTTTTCGGTCGGATTTGGTCGTTGAGTGGTTTTTTGAGTTGTTTTAAGGATGTAGAGGATTTTATTTTTATTGTTTATTGGGGGTTATTTTGAGTCGGGTTGCGTTTTGGGTTGGCGGCGAG.

[0098] Furthermore, the underlined portion in the converted DNA chain sequence is the position of the methylation primer probe sequence of TCTEX1D1 (chr1: 67217979-67218393) finally obtained.

[0099] Furthermore, in the sequence of the converted DNA chain in the methylated region sequence of the gene TCTEX1D1, all C in CG in the converted DNA chain sequence is not methylated, and bisulfite conversion is performed, and the unmethylated C is converted to T, and the methylated C remains unchanged, thereby obtaining a converted DNA chain; further, the sequence of the converted DNA chain is as follows:

[0100] GGAGAGGAGGAATAAGGTGGTGGAGAGATTGGGGTTAAGTAGGGTTATTTTTTAATAAAGGGTTGATTTTTTGTTTTTTATTTTTTTTTTGGGGTTTAGAT GTGTTGTAGAGAGAGGTGATTT TATTT GTAGTAGGGAGGTTTT GTTTGGAGTTTT TTAGAT TTTTGGTATTGTAGATATTGTGTGT GGTGTGGTTTTTGTGTTTTAGGTTTTATTTAGTTGGTGGTTGTTTGTGTATTTTTTTTGGATTTTGAGGTTGGTTTTTAGGGTGATTTTAAATAGAGTTTTTGGTTTTTTGGTTGGATTTGGTTGTTGAGTGGTTTTTTGAGTTGTTTTAAGGATGTAGAGGATTTTATTTTTATTGTTTATTGGGGGTTATTTTGAGTTGGGTTGTGTTTTGGGTTGGTGGTGAG.

[0101] Furthermore, the underlined portion in the converted DNA chain sequence is the position of the unmethylated primer probe sequence of TCTEX1D1 (chr1: 67217979-67218393) obtained in the end.

[0102] Furthermore, the PCR reaction procedure of the ZNF300 gene region is as follows:

[0103]

[0104]

[0105] Furthermore, the methylation region sequence of the gene ZNF300 is selected from the DNA strand in UCSC Genome Browser on Human (GRCh37 / hg19), hg19_dna range=chr5:150284285-1502847355'pad=03'pad=0strand=+; the sequence of the DNA strand is as follows:

[0106] GCCTCTGACTCCCAGCTCGATTATCAGGTCCCAAAACCACCATCACCGCTTAATGGGAACCGCTGTCACATCCCTATTGGCAAACTCTCCTGCTTCCTGTCCGCGAGCACCACACCGGGCTCCCGAGGCTGCGCCGTTCCGTACTGGGCGGTTTTGCCCGTTCCGCATCTTCAGAGGCTTTGTTCAGGAAGCAACATGGCTGCTCCCTGGAGCTGTTTCCGCATGG GGCCGCCATCTTGAGAGCCCGGTCGTCTTCGTACTAGCGGAGGATTCGCCCAGTGCTGAGCCCGAGAATGGGCTGTACCACAGCGGCAGCGAAGGGAGGGGAAGGCGACGGAAAGGAGGCCTGCGAAAGGAGCATTAAGGAGCACACTTTTGCAGAAAGATTTTACAAGGGGGATTGAAGAGGAAGTTGGAGTGAACCTTCCTCTATTGGAAGCATGCTTCTTGAC.

[0107] Furthermore, all C in the CG in the DNA chain sequence in the methylated region sequence of the gene ZNF300 are methylated and subjected to bisulfite conversion, wherein the unmethylated C is converted to T and the methylated C remains unchanged, thereby obtaining a converted DNA chain. Furthermore, the sequence of the converted DNA chain is as follows:

[0108] GTTTTTGATTTTTAGTTCGATTATTAGGTTTTAAAATTATTATTATCGTTTAATGGGAATCGTTGTTATATTTTATTGGTAAATTTTTTTGTTTTTTGTTCGCGAGTATTATATCGGGTTTTCGAGGTTGCGTCGTTTCGTATTGGGCGGTTTTGTTCGTTTCGTATTTTTAGAGGTTTTGTTTAGGAAGTAATATGGTTGTTTTTTGGAGTTGTTTT C GTATGGGGTCGTTATTTTGAGAG TTCGGTCGTTTTCGT ATTAGCGGAGGATTCGTTTAGTGTTGAGTT CGAGAATGG GTTGTATTATAGCGGTAGCGAAGG GAGGGGAAGGCGACGGAAAGGAGGTTTGCGAAAGGAGTATTAAGGAGTATATTTTTGTAGAAAGATTTTATAAGGGGGATTGAAGAGGAAGTTGGAGTGAATTTTTTTTATTGGAAGTATGTTTTTGAT.

[0109] Furthermore, the underlined portion in the converted DNA chain sequence is the position of the methylation primer probe sequence of ZNF300 (chr5: 150284285-150284735) finally obtained.

[0110] Furthermore, in the sequence of the converted DNA chain in the methylated region sequence of the gene ZNF300, all C in CG in the converted DNA chain sequence is not methylated, and bisulfite conversion is performed, and the unmethylated C is converted to T, and the methylated C remains unchanged, thereby obtaining a converted DNA chain; further, the sequence of the converted DNA chain is as follows:

[0111] GTTTTTGATTTTTAGTTTGATTATTAGGTTTTAAAATTATTATTGTTTAATGGGAATTGTTGTTATATTTTTATTGGTAAATTTTTTTGTTTTTTGTTTGTGAGTATTATTGGGTTTTTGAGGTTGTGTTGTTTTGTATTGGGTGGTTTTGTTTGTTTTGTATTTTTAGAGGTTTTGTTTAGGAAGTAATATGGTTGTTTTTTGGAGTTGT TTTT GTATGGGGTTGTTATTTTGA GAGTTTGGTTGTTTTTGT ATTAGTGGAGGATTTGTTTAGTGTTGAGTT TGAGAATGGG TTGTATTATAGTGGTAGTGAAGGG AGGGGAAGGTGATGGAAAGGAGGTTTGTGAAAGGAGTATTAAGGAGTATATTTTTGTAGAAAGATTTTATAAGGGGGATTGAAGAGGAAGTTGGAGTGAATTTTTTTTATTGGAAGTATGTTTTTGAT.

[0112] Furthermore, the underlined portion in the converted DNA chain sequence is the position of the unmethylated primer probe sequence of ZNF300 (chr5: 150284285-150284735) obtained in the end.

[0113] Furthermore, the base sequences of the front and back primers and probe of the internal reference GAPDH gene region are as follows:

[0114]

[0115]

[0116] Furthermore, the PCR reaction procedure of the internal reference GAPDH gene region is as follows:

[0117]

[0118] Further, the methylation region sequence of the internal reference gene GAPDH is selected from the DNA strand in UCSC Genome Browser on Human (GRCh37 / hg19), hg19_dna range=chr12:6644961-66451685'pad=03'pad=0strand=+; the sequence of the DNA strand is as follows:

[0119] CACCCTGGTCTGAGGTTAAATATAGCTGCTGACCTTTCTGTAGCTGGGGGCCTGGGCTGGGGCTCTCTCCCATCCCTTCTCCCCACACACATGCACTTACCTGTGCTCCCACTCCTGATTTCTGGAAAAGAGCTAGGAAGGACAGGCAACTTGGCAAATCAAAGCCCTGGGACTAGGGGGTTAAAATACAGCTTCCCCTCTTCCCACC.

[0120] Furthermore, all C in the CG in the sequence of the DNA chain in the methylation region sequence of the internal reference gene GAPDH is methylated, and bisulfite conversion is performed, wherein the unmethylated C is converted to T, and the methylated C remains unchanged, to obtain a converted DNA chain. Furthermore, the sequence of the converted DNA chain is as follows:

[0121] TATTTTGGTTT GAGGTTAAATATAGTTGTTGA TTTTTTGTAGTTGGGGGTTTGGGTTGGGGTTTTTTTTTTTTTTTTTTTATATATATGTATTTATTTGTGTTTTTTTTTGATTTTTGGAAA AGAGTTAGGAAGGATA GGT AATT TGGTAAATTAAAGTTTTGGG ATTAGGGGGTTAAAATATAGTTTTTTTTTTTTTTATT.

[0122] Furthermore, the underlined portion of the converted DNA chain sequence is the position of the methylation primer probe sequence of GAPDH (chr12: 6644961-6645168) obtained in the end. Furthermore, in the present invention, a digital PCR platform is used to design two methods to test the methylation rate of the gene ( Figure 1 ), the first is the primer probe test of methylation (M) and unmethylation (NM), wherein the gene methylation rate M1P = copy number (gene_M) / (copy number (gene_UM)+copy number (gene_M)); the second is the primer probe test of methylation (gene_M) and internal reference gene (IC), wherein the methylation rate M2P = copy number (gene_M) / copy number (IC).

[0123] Furthermore, the primers and / or probes for detecting gene methylation described in the present invention are selected from the regions chr20:2781212-2781649, chr6:10881846-10882051, chr11:31848487-31848976, chr10:22634000-22634862, chr1:67218079-67218293, chr5:150284385-150284635 of the target genes CPXM1, GCM2, PAUPAR, SPAG6, TCTEX1D1, and ZNF300, and each region can be detected individually or in combination for diagnosis.

[0124] In a second aspect, the present invention provides a methylation detection kit for diagnosing breast cancer, comprising at least one set of the above-mentioned region-specific primers and / or probes.

[0125] A method for diagnosing breast cancer using the above-mentioned region-specific primers and probes of the present invention comprises the following steps:

[0126] Step (1): plasma / serum free DNA extraction and sulfite conversion;

[0127] Step (2): Digital PCR platform gene methylation test;

[0128] Step (3): Interpretation of gene methylation results.

[0129] In a third aspect, the present invention also provides the use of the above-mentioned region-specific primers and probes in the preparation of a kit for detecting breast cancer; the above-mentioned region-specific primers and probes can be used for detection alone or in combination.

[0130] Compared with the prior art, the present invention has the following beneficial effects:

[0131] (1) The present invention uses an automated high-throughput instrument to detect the methylation level of tumor suppressor genes in free circulating tumor DNA (ctDNA) in peripheral blood plasma or serum to screen for breast cancer. This method uses non-invasive sampling, can be monitored repeatedly, and can be detected automatically.

[0132] (2) The method of the present invention has the characteristics of high sensitivity (>80%) and good specificity (>85%).

[0133] (3) The method of the present invention is suitable for large-scale screening of breast cancer, and provides a new technical means for screening of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0134] Figure 1 Schematic diagram of digital PCR MethyLight.

[0135] Figure 2 The distribution of gene methylation rate in breast cancer and benign lesions.

[0136] Figure 3 The area under the receiver operating characteristic curve (AUC) for the detection of M1P and M2P methylation of CPMX1 for the diagnosis of breast cancer.

[0137] Figure 4 The area under the receiver operating characteristic curve (AUC) for the detection of M1P and M2P methylation of GCM2 for the diagnosis of breast cancer.

[0138] Figure 5 The area under the receiver operating characteristic curve (AUC) for the detection of M1P and M2P methylation of PAUPAR for the diagnosis of breast cancer.

[0139] Figure 6 The area under the receiver operating characteristic curve (AUC) for SPAG6 M1P and M2P methylation detection in diagnosing breast cancer.

[0140] Figure 7 The area under the receiver operating characteristic curve (AUC) for the detection of M1P and M2P methylation of TCTEX1D1 for the diagnosis of breast cancer.

[0141] Figure 8 The area under the receiver operating characteristic curve (AUC) for the detection of ZNF300 M1P and M2P methylation for the diagnosis of breast cancer. DETAILED DESCRIPTION

[0142] For ease of understanding, the application will be described more fully below, and preferred embodiments of the application are provided. However, the application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In the embodiment, if specific techniques or conditions are not indicated, the techniques or conditions described in the document in this area or according to the product specification are carried out. Reagents used or instruments that do not indicate manufacturers are conventional products that can be obtained through the market.

[0143] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0144] Test methods

[0145] 1. Plasma / serum free DNA extraction and sulfite conversion

[0146] pass Genomic DNA was extracted from stored plasma / serum samples using the DNAMini Kit (Qiagen, Venlo, Netherlands). A total of 25 μL of DNA was obtained from 250 μL. After DNA extraction, 22.5 μL of plasma / serum DNA was immediately subjected to sulfite conversion using the EZ DNA Methylation Kit (Zymo Research, California, US). After multiple mixing, centrifugation, and 2 water baths, the M-dilution buffer was added and incubated at 37°C for 15 minutes, and the CT conversion reagent was added and incubated at 50°C for 12-16 hours, 10 μL of sulfite-converted DNA was finally obtained. With the methylation kit, methylated cytosine (C) remains unchanged, while unmethylated cytosine residues (C) are converted to uracil (U) and recognized as thymine (T) in the PCR amplification reaction.

[0147] 2. Digital PCR platform gene methylation test

[0148] 1. Prepare PCR reaction solution

[0149] The DNA treated with sulfite was immediately subjected to gene methylation detection in the QX200 ddPCR system (BioRad, California, US). The total volume of the ddPCR reaction system was 25 μL, consisting of 8 μL of converted plasma / serum DNA, 12.5 μL of 1×ddPCR Supermix (Bio-Rad), primers, and probes with final concentrations of 800 nM and 400 nM, respectively. The final concentrations of primers and probes were 200-1200 nM and 100-800 nM, respectively. The sequences of primers and probes are shown in the table "Base sequences of front and back primers and probes for detecting each methylation target gene region".

[0150] 2. Preparation of microdroplets

[0151] Prepare droplets in a QX200 droplet generator (Bio-Rad). Unpack the 8-well droplet generation plate and install it on the bracket. Note that the direction of the droplet generation plate is consistent with the direction marked on the bracket. Add 20ul of the reaction system to the 8 wells in the middle row of the droplet generation plate. If there are less than 8 samples, add 20ul of 1× buffer control diluted once to make up for it. Do not leave any empty holes. Add 70ul of droplet generation oil (DG Oil) to each of the 8 wells in the bottom row of the droplet generation plate. Do not leave any empty holes. Cover with a rubber pad, making sure that the small holes on both sides are hooked firmly; place it gently and steadily in the droplet generation instrument, start generating droplets, and pay attention to the indicator light status on the instrument. It usually takes about 2 minutes to complete. After completion, droplets are generated in the top row of wells on the plate.

[0152] 3. Perform PCR amplification

[0153] Use a pipette to carefully draw 40 μl from the droplet generation plate and slowly transfer it to the 96-well PCR plate. Since digital PCR detects whether each droplet contains fluorescence in the end, thereby determining whether the droplet contains a template, rather than real-time observation of the number of cycles of fluorescence changes in each droplet or tube, the QX200 ddPCR system C100 Touch thermal cycler is used for digital PCR amplification. The amplification parameters are determined according to the primers and probes, see the primer and probe table of each gene.

[0154] 4. Read and analyze the results

[0155] After the PCR amplification is completed, different numbers of droplets in each well will generate fluorescent signals. Next, you need to read these droplets and count the total number of droplets in each well and the number of droplets containing fluorescence. Put the 96-well plate that has completed the PCR into the base, cover the top bracket, and then gently and steadily put it into the droplet reader (QX200 TM DropletReader), and gene methylation data were analyzed using QuantaSof Analysis Pro software (Bio-Rad). Figure 1 The two formulas in can obtain the two methylation rates of the gene.

[0156] 3. Interpretation of gene methylation results

[0157] 1. Counting of gene methylation rate

[0158] Digital PCR directly counts the number of DNA molecules in each PCR reaction. The gene methylation (M) probe counts the methylated copy number, the gene unmethylation (UM) probe counts the unmethylated copy number, and the internal reference (IC) gene probe counts the total copy number. The counting formula for the gene methylation rate is:

[0159]

[0160] 2. Interpretation of the results of gene methylation markers in diagnosing breast cancer (see the table below)

[0161] Interpretation criteria for gene methylation diagnosis of breast cancer

[0162]

[0163]

[0164] Example 1

[0165] Blood samples from 56 patients with clear pathological results were collected, including 28 benign lesions and 28 breast cancers, of which 2 were male breast invasive ductal carcinomas. According to molecular classification, there were 6 luminal A type, 10 luminal B type, 10 her2 high expression type, and 2 triple negative type breast cancers.

[0166] The gene methylation test was completed according to Example 1. When the external quality control was qualified, the results of the methylation rates of CPMX1, GCM2, PAUPAR, SPAG6, TCTEX1D1, and ZNF300 genes in two ways were obtained for each sample. The distribution of gene methylation rates in breast cancer and benign lesions is different, see Figure 2 Through receiver operating characteristic curve (ROC) analysis, the difference between the two methods in the ability of the gene of the present invention to identify breast cancer was not statistically significant. The specific results are as follows Figures 3 - 8 shown.

[0167] According to the maximum Youden index principle, the critical values ​​of CPMX1, GCM2, PAUPAR, SPAG6, TCTEX1D1, and ZNF300 gene methylation were C1k1=0.0465, C2k1=0.061, G1k2=0.0595, G2k2=0.0435, P1k3=0.0500, P2k3=0.0435, S1k4=0.0585, S2k4=0545, T1k5=0.0540, T2k5=0.0500, Z1k6=0.0620, and Z2k6=0.0655. The clinical sensitivity and specificity of CPMX1, GCM2, PAUPAR, SPAG6, TCTEX1D1, and ZNF300 gene methylation in diagnosing breast cancer were all above 80%, as shown in the table below. Among them, the consistency rate of sensitivity and specificity of the two test methods for gene methylation detection was 75% (9 / 12) when different critical values ​​were taken.

[0168] AUC, sensitivity and specificity of gene methylation in diagnosing breast cancer

[0169]

[0170] The breast cancer gene methylation marker of the present invention has complementary function in detecting breast cancer. For example, 3 breast cancer cases with negative GCM2 detection were positive by SPAG6. And 4 breast cancer cases with negative SPAG6 detection were positive by GCM2. The results are as follows:

[0171] Results of gene methylation (M1P) detection in breast cancer patient samples

[0172]

[0173]

[0174]

[0175] The above-mentioned embodiments only express the implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims

1. A primer and probe set for detecting specific methylation levels of breast cancer, characterized in that: The target genes and corresponding chromosome regions are shown in the following table: ； Wherein, the methylation region sequence of the above gene is selected from GRCh37 / hg19; Primers or probes were designed for the above target genes and regions. The sequences of the primers and probes are as follows: Forward primer sequences for CPXM1 gene: TAGTTCGTTGTTCGTTTCGGATTTCG and TTGTTTGTTTTGGATTTTGGAGAGGA; The latter primer sequences for the CPXM1 gene were: ACAAAATCGACGATCCTAAAACTTAAAACG and AATCAACAATCCTAAAACTTAAAACAACCC; Probe sequences for the CPXM1 gene: FAM-AAAATCAACCCCGCTCGCTCCTCCCG and HEX-AAATCAACCCCACTCACTCCTCCCAACCCT; Forward primer sequences for the GCM2 gene: GTTTCGCGTGTTCGTATATATTCG and GTTTTGTGTGTTTGTATATATTTGGT; The sequences of the rear primers for the GCM2 gene were: TATACTCCTACGAAATACAACTCAACT and ATATACTCCTACAAAATACAACTCAAC; Probe sequences for the GCM2 gene: FAM-AAACATCAACGATCCGCAAATACCTCAAAT and HEX-AAACATCAACAATCCACAAATACCTCAAAT; Forward primer sequences for PAUPAR gene: GGGGTTTATAGCGGGTAGTCG and GGGGTTTATAGTGGGTAGTTGAG; The sequences of the rear primers for the PAUPAR gene: CGAATCAACGACCGAACACCAACC and CAAATCAAACCAAACACCAACCTA; Probe sequences for the PAUPAR gene: FAM-CTCCCAAACTCCGAATCCGCCAACC and HEX-ACCTCCCAAACTCCAAATCCACCAACC; Forward primer sequences for SPAG6 gene: ATTACGGTAATTTGGGGTCGTT and TTAGGATATTATGGTAATTTGGGGTTG; The sequences of the rear primers for the SPAG6 gene: CGCGAAACCGCCGAAAC and CCAAACTCACAAAACCACCAAA; Probe sequences for the SPAG6 gene: FAM-CGCCGTCGAATATCCCTACACTACCCG and HEX-CGCCGTCGAATATCCCTACACTACCCG; Forward primer sequences for TCTEX1D1 gene: GCGTTGTAGAGAGAGGCGAT and GTGTTGTAGAGAGAGGTGATTT; The sequences of the rear primers for the TCTEX1D1 gene were: CGCCAATATCTACAATACCGA and ACACACAATATCTACAATACCAAAA; Probe sequences for the TCTEX1D1 gene: FAM-AAACTCCGAACGAAACCTCCCTACTAC and HEX-AAAACTCCAAACAAAACCTCCCTACTAC; Forward primer sequences for the ZNF300 gene: CGTATGGGGTCGTTATTTTGAGAG and TTTTGTATGGGGTTGTTATTTTGA; The sequences of the rear primers for the ZNF300 gene were CCTTCGCTACCGCTATAATACAAC and CCCTTCACTACCACTATAATACAA; Probe sequences for the ZNF300 gene: FAM-AACTCAACACTAAACGAATCCTCCGCTAAT and HEX-AACTCAACACTAAACAAATCCTCCACTAAT; Forward primer sequence of GAPDH gene: GAGGTTAAATATAGTTGTTGA; The rear primer sequence of GAPDH gene: CCCAAAACTTTAATTTACCA; Probe sequence for GAPDH gene: HEX-ACCT <lna>ATC <lna>CTT <lna>CCT <lna> AACTCT.< / lna> < / lna> < / lna> < / lna> 2. A kit for detecting methylation level of breast cancer, characterized in that: The invention comprises the primer and probe set for detecting the specific methylation level of breast cancer as described in claim 1.

3. Use of the primer and probe set for detecting the specific methylation level of breast cancer according to claim 1 in preparing a kit for detecting the methylation level of breast cancer.

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