WO2021185275A1 - Probe composition for detecting 11 cancers - Google Patents

Probe composition for detecting 11 cancers Download PDF

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Publication number
WO2021185275A1
WO2021185275A1 PCT/CN2021/081276 CN2021081276W WO2021185275A1 WO 2021185275 A1 WO2021185275 A1 WO 2021185275A1 CN 2021081276 W CN2021081276 W CN 2021081276W WO 2021185275 A1 WO2021185275 A1 WO 2021185275A1
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seq
cancer
probe
tissue
specific
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PCT/CN2021/081276
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French (fr)
Chinese (zh)
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韩晓亮
李永君
吴宁宁
郭媛媛
王建铭
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博尔诚（北京）科技有限公司
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Priority to JP2022521064A priority Critical patent/JP7462035B2/en
Priority to CN202180021008.4A priority patent/CN115279924A/en
Publication of WO2021185275A1 publication Critical patent/WO2021185275A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • This application relates to a cancer gene methylation detection composition, in particular to a probe composition that specifically recognizes the DNA sequence after Bisulfite treatment, and detection based on high-throughput sequencing (NGS) method
  • NGS high-throughput sequencing
  • NGS High-throughput sequencing
  • This technology can simultaneously sequence dozens to millions of DNA molecules, marking the arrival of the post-genome era.
  • different goals such as de novo sequencing and resequencing can be achieved, and the sequence of the genome, transcriptome, and methylome can also be analyzed through different pre-processing.
  • PCR polymerase chain reaction
  • FISH fluorescence in situ hybridization
  • gene chip technology has low price, high sensitivity, simple and fast operation, and high clinical popularity.
  • FISH fluorescence in situ hybridization
  • the gene chip throughput is higher than the former two, and it can detect a large number of genes at the same time.
  • the limitation is that it can only detect known genes or mutations, with low accuracy and high false positives.
  • the NGS technology has the characteristics of high throughput (detecting a large number of known and unknown genes and mutations at the same time), accurate results (higher accuracy than gene chips), fast detection speed, and low cost of detection for each gene. Now Has been gradually applied to clinical disease detection and monitoring and other fields. With the further reduction of sequencing costs in the future, NGS will inevitably gradually replace other high-throughput technologies such as gene chips.
  • target sequence capture and sequencing has become a more mainstream choice.
  • This technology is based on detection requirements, designing capture probes for the genomic region of interest, enriching the target fragment DNA through the principle of hybridization and complementation, and subsequently performing NGS detection.
  • This strategy can be flexibly customized according to the purpose of research or detection, selecting only a small number of gene regions, increasing the depth of sequencing, and effectively discovering the mutation status of the target region, with high sensitivity and accuracy.
  • Liquid biopsy is a method of in vitro diagnosis. It uses non-invasive blood testing to monitor circulating tumor cells (CTC) or circulating tumor DNA (ctDNA) released into the blood by tumors or metastases. This technology can effectively reduce invasiveness. It can realize the sampling of all parts of the tumor and all metastases, overcome tumor heterogeneity (and the current standard tissue biopsy can only reflect the characteristics of a certain part of the tumor), and realize real-time monitoring with higher sensitivity , It is even possible to predict the location of the lesion based on genomic information, which can effectively prolong the survival time of patients. Based on these advantages, liquid biopsy can be used for early diagnosis of tumors, auxiliary staging, prognosis and recurrence monitoring, medication guidance and other aspects. Currently the most commonly used liquid biopsy of free DNA.
  • CtDNA Cell-free DNA
  • cfDNA is a partially degraded endogenous DNA that exists in circulating blood and is free and extracellular. Studies have shown that in the process of tumor tissue development, after tumor cell apoptosis, DNA will be released into plasma, and after degradation, free tumor DNA (ctDNA) will be formed.
  • the molecular genetic characteristics of CtDNA (such as gene mutation, microsatellite instability, and tumor suppressor gene promoter methylation, etc.) are consistent with tumor tissue DNA.
  • the collection of peripheral blood is simpler than other clinical detection methods, easy to promote to the grassroots, and because of its non-invasive characteristics, it is easier to be accepted by asymptomatic people. Therefore, the detection of changes in the level of ctDNA methylation in plasma can become one of the important methods for early screening and diagnosis of multiple cancers.
  • Using target sequence capture technology combined with NGS to monitor cfDNA variation and methylation level changes can realize early tumor screening, susceptibility gene monitoring, companion diagnosis, personalized medication, prognostic monitoring and other applications.
  • many companies at home and abroad have launched cancer detection panels of different scales for different application scenarios. Some panels have obtained FDA or CFDA approval numbers.
  • FoundationOne CDx launched by Foundation Medicine covers 324 genes
  • IMPACT launched by Memorial Sloan Kettering Cancer Research Center (MSK) covers 468 cancer-related genes
  • Burning Rock Medicine launched "human EGFR/ALK/BRAF/KRAS gene mutations” Joint detection kit”, “Human EGFR, KRAS, BRAF, PIK3CA, ALK, ROS1 gene mutation detection kit” launched by Nuohe Zhiyuan, etc.
  • Kunyuan Gene also launched the product "Chang Lesi” to detect the methylation level of colorectal cancer in 2018.
  • this application provides a probe composition that can be used for esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, and breast cancer.
  • a probe composition can be used for esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, and breast cancer.
  • the probe composition can: 1) be used in a non-invasive way for early screening of asymptomatic people and prognostic detection of cancer patients, reducing the harm caused by invasive detection, 2) increasing the depth of sequencing and making
  • the breadth of gene detection is better than existing technologies and products, with high throughput, faster detection speed, and low cost of detection for each gene.
  • this application involves the following:
  • a probe composition comprising: targeting any cancer among esophageal cancer, stomach cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer
  • the specific region of the probe, wherein the cancer-specific region is selected from any of Seq ID No.: 1-62.
  • the probe composition according to item 1 further comprising: any probe targeting a pan-cancer specific region, the pan-cancer specific region selected from Seq ID No.: 63-64 Arbitrarily.
  • the probe composition according to item 3 characterized in that the Seq ID No.: 66-67, 79, 81-82, 97-102, 116-117 in the tissue-specific region is esophageal Tissue specific target area.
  • the probe composition according to item 3 characterized in that Seq ID Nos.: 81, 82, 97-102 in the tissue-specific region are tissue-specific target regions of the stomach.
  • the probe composition according to item 3 characterized in that Seq ID No. 83, 87-90, 110 in the tissue-specific region are tissue-specific target regions of the colorectal.
  • the probe composition according to item 3 characterized in that Seq ID No. 65, 70-72, 76, 91-92, 103-104 in the tissue-specific region are tissue-specific of the lung sexual target area.
  • tissue-specific regions Seq ID No.: 73-74 and 107 are tissue-specific target regions of the liver.
  • the probe composition according to item 3 characterized in that Seq ID No. 80, 84, 93, 95 in the tissue-specific region are tissue-specific target regions of the prostate.
  • the probe composition according to item 3 characterized in that Seq ID Nos.: 78 and 84-86 in the tissue-specific region are tissue-specific target regions of the breast.
  • tissue-specific region Seq ID No.: 77, 96, and 105 are tissue-specific target regions of the ovary.
  • the probe composition according to item 3 characterized in that Seq ID No.: 68-69, 75, 109 in the tissue-specific region are tissue-specific target regions of the cervix.
  • the probe composition according to item 3 characterized in that Seq ID Nos. 94, 106, 108 in the tissue-specific region are tissue-specific target regions of the endometrium.
  • the probe composition according to any one of items 1-14 comprising: a hypomethylation probe, which is compatible with the bisulfite-converted CG-free CG methylation specificity Regional hybridization, and; a hypermethylated probe, which hybridizes to the specific region where the bisulfite-converted CG is fully methylated.
  • each probe has a length of 45-56 bp, preferably 50-56 bp, and more preferably 50 bp.
  • hypomethylation probe comprises a probe targeting a cancer-specific region.
  • Seq ID No. any of 118-214
  • the hypermethylated probe includes a probe targeting a cancer-specific region Seq ID No.: any of 275-371, targeting pan-cancer Seq ID No. of probes for specific regions: any of 372-373, and Seq ID No. of probes for tissue-specific regions: any of 374-431.
  • a kit comprising the probe composition according to any one of items 1-19.
  • a probe composition according to any one of items 1-19 is prepared for detecting esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, and cervical cancer And the application of the kit for endometrial cancer.
  • the application also provides a method for detecting 11 kinds of esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer by using the probe composition Methods of cancer.
  • This application also provides a method for simultaneously detecting changes in the methylation level of the 11 cancers mentioned above by using the kit.
  • Liquid biopsy is a non-invasive tumor detection, which can be applied to asymptomatic and patient groups who cannot obtain tissue samples.
  • the average sequencing depth exceeds 5000X.
  • the screening of all high-incidence cancers can be completed at one time, which improves the detection efficiency.
  • the average price of each marker is lower than the existing single-marker detection in the market.
  • FIG 1 is the implementation process of this application
  • This application provides a probe composition for cancer gene methylation detection.
  • NGS high-throughput sequencing
  • the probe is a single-stranded or double-stranded DNA with a length of tens to hundreds or even thousands of base pairs, which can take advantage of molecular denaturation, renaturation and high accuracy of base complementary pairing, and can be complementary to the sample to be tested
  • the unlabeled single-stranded DNA or RNA is hydrogen-bonded (hybridized) to form a double-stranded complex (hybrid).
  • detection systems such as autoradiography or enzyme-linked reaction can be used to detect the results of the hybridization reaction.
  • the region that complementarily binds or hybridizes with the probe is the specific target region. Multiple probes are combined into a probe composition.
  • a cancer-specific region refers to a significant difference in the methylation level of this region compared with normal control tissues in a small number of cancer types.
  • pan-cancer specific region refers to a significant difference in the methylation level of this region compared with normal control tissues in most cancer types.
  • Tissue-specific region refers to the significant difference in the methylation level of the region in a specific tissue compared with other tissues.
  • DNA methylation refers to the methylation process that occurs at the 5th carbon atom of cytosine in CpG dinucleotides. As a stable modification state, DNA methyltransferase can follow DNA The duplication process of dna is inherited to the new generation DNA, which is an important epigenetic mechanism. When DNA is methylated, the methylation of the gene promoter region can lead to the silence of tumor suppressor gene transcription, so it is related to the occurrence of tumors. close. Abnormal methylation includes hypermethylation of tumor suppressor genes and DNA repair genes, hypomethylation of repetitive sequence DNA, and loss of imprinting of certain genes, which are related to the occurrence of a variety of tumors.
  • Panel refers to the probe composition used in this article.
  • a probe composition includes: targeted esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and A probe for a specific region of any cancer in endometrial cancer, wherein the cancer-specific region is selected from any of Seq ID No.: 1-62; a probe that targets any of the pan-cancer specific regions Needle, the pan-cancer-specific region is selected from Seq ID No.: any of 63-64; and any probes that target tissue-specific regions, the tissue-specific region is selected from Seq ID No.: Any of 65-117.
  • Seq ID Nos.: 66-67, 79, 81-82, 97-102, 116-117 of the tissue-specific regions are tissue-specific target regions of the esophagus
  • Seq ID No.: 83, 87-90, 110 are the tissue-specific target area of the colorectal.
  • a probe composition includes: targeted esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and A probe for a specific region of any cancer in endometrial cancer, wherein the cancer-specific region is selected from Seq ID No.: any of 1-62; a probe that targets any of the pan-cancer specific regions Needle, the pan-cancer-specific region is selected from Seq ID No.: any of 63-64; and any probes that target tissue-specific regions, the tissue-specific region is selected from Seq ID No.: Any of 65-117. Seq ID No. of the tissue-specific regions: 65, 70-72, 76, 91-92, 103-104 are the tissue-specific target regions of the lung.
  • a probe composition includes: targeted esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and A probe for a specific region of any cancer in endometrial cancer, wherein the cancer-specific region is selected from Seq ID No.: any of 1-62; a probe that targets any of the pan-cancer specific regions Needle, the pan-cancer-specific region is selected from Seq ID No.: any of 63-64; and any probes that target tissue-specific regions, the tissue-specific region is selected from Seq ID No.: Any of 65-117.
  • Seq ID No.: 73-74 and 107 are tissue-specific target regions of the liver
  • Seq ID No.: 111-115 are tissue-specific target regions of the pancreas.
  • a probe composition includes: targeted esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and A probe for a specific region of any cancer in endometrial cancer, wherein the cancer-specific region is selected from Seq ID No.: any of 1-62; a probe that targets any of the pan-cancer specific regions Needle, the pan-cancer-specific region is selected from Seq ID No.: any of 63-64; and any probes that target tissue-specific regions, the tissue-specific region is selected from Seq ID No.: Any of 65-117. Seq ID No. in the tissue-specific region: 80, 84, 93, and 95 are tissue-specific target regions of the prostate.
  • a probe composition includes: targeted esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and A probe for a specific region of any cancer in endometrial cancer, wherein the cancer-specific region is selected from Seq ID No.: any of 1-62; a probe that targets any of the pan-cancer specific regions Needle, the pan-cancer-specific region is selected from Seq ID No.: any of 63-64; and any probes that target tissue-specific regions, the tissue-specific region is selected from Seq ID No.: Any of 65-117.
  • Seq ID No.: 78, 84-86 are the tissue-specific target regions of the breast
  • Seq ID No.: 77, 96, and 105 are the tissue-specific target regions of the ovary
  • Seq ID No.: 68-69, 75, and 109 are the tissue-specific target regions of the cervix
  • Seq ID No.: 94, 106, and 108 are the tissue-specific target regions of the endometrium.
  • the above-mentioned probe composition includes a hypomethylated probe which hybridizes to the bisulfite-converted specific region without CG methylation, and high methylation A probe that hybridizes to the specific region where the bisulfite-converted CG is fully methylated.
  • the hypomethylation probes include probes targeting cancer-specific regions Seq ID No.: any of 118-214, and probes targeting pan-cancer specific regions Seq ID No.: 215-216 Any, and the probe Seq ID No. that targets tissue-specific regions: Any of 217-274.
  • the hypermethylation probes include probes targeting cancer-specific regions, Seq ID No.: any of 275-371, and probes targeting pan-cancer specific regions, Seq ID No.: 372-373, Seq ID No.: any of 374-431 probes targeting tissue-specific regions.
  • Seq ID No. 118, Seq ID No. 119 and Seq ID No. 120 are all hypomethylated probes that target the target region shown in Seq ID No. 1
  • Seq ID No. 275 , Seq ID No. 276 and Seq ID No. 277 are all hypermethylated probes that target the target region shown in Seq ID No. 1.
  • Seq ID No. 121 and Seq ID No. 122 are hypomethylation probes that target the target region shown in Seq ID No. 2
  • Seq ID No. 278 and Seq ID No. 279 are both targeted to Seq ID No. .2 Hypermethylated probes in the target region shown.
  • Seq ID No. 124 are hypomethylation probes that target the target region shown in Seq ID No. 3, and Seq ID No. 280 and Seq ID No. 281 are both targeted to Seq ID No. .3 Hypermethylated probes in the target region shown.
  • Seq ID No. 125 is a hypomethylated probe that targets the target region shown in Seq ID No. 4, and Seq ID No. 282 is a hypermethylated probe that targets the target region shown in Seq ID No. 4.
  • Seq ID No. 126 and Seq ID No. 127 are both hypomethylated probes that target the target region shown in Seq ID No. 5, and Seq ID No. 283 and Seq ID No. 284 are both targeted to Seq ID No. .3 Hypermethylated probes in the target region shown.
  • Seq ID No. 128 is a hypomethylated probe targeting the target region shown in Seq ID No. 6, and Seq ID No. 285 is a hypermethylated probe targeting the target region shown in Seq ID No. 6.
  • Seq ID No. 129 is a hypomethylated probe targeting the target region shown in Seq ID No. 7, and Seq ID No. 286 is a hypermethylated probe targeting the target region shown in Seq ID No. 7.
  • Seq ID No. 130 and Seq ID No. 131 are both hypomethylated probes that target the target region shown in Seq ID No. 8, and Seq ID No. 287 and Seq ID No. 288 are both targeted to Seq ID No.
  • Seq ID No. 133 and Seq ID No. 134 are all hypomethylated probes that target the target region shown in Seq ID No. 9, Seq ID No. 289, Seq ID No. 290 and Seq ID No. 291 is a hypermethylated probe that targets the target region shown in Seq ID No. 9.
  • Seq ID No.135 is a hypomethylated probe targeting the target region shown in Seq ID No.10
  • Seq ID No.292 is a hypermethylated probe targeting the target region shown in Seq ID No.10.
  • Seq ID No.136 is a hypomethylated probe targeting the target region shown in Seq ID No.11
  • Seq ID No.293 is a hypermethylated probe targeting the target region shown in Seq ID No.11.
  • Seq ID No.137 is a hypomethylated probe targeting the target region shown in Seq ID No.12
  • Seq ID No.294 is a hypermethylated probe targeting the target region shown in Seq ID No.12.
  • Seq ID No.138 is a hypomethylated probe targeting the target region shown in Seq ID No.13
  • Seq ID No.295 is a hypermethylated probe targeting the target region shown in Seq ID No.13.
  • Seq ID No. 139 and Seq ID No. 140 are both hypomethylated probes that target the target region shown in Seq ID No. 14, and Seq ID No. 296 and Seq ID No. 297 are both targeted to Seq ID No.
  • Seq ID No. 141 and Seq ID No. 142 are hypomethylated probes that target the target region shown in Seq ID No. 15, and Seq ID No. 298 and Seq ID No. 299 are both targeted to Seq ID No.
  • Seq ID No. 143 and Seq ID No. 144 are hypomethylated probes that target the target region shown in Seq ID No. 16, and Seq ID No. 300 and Seq ID No. 301 are both targeted to Seq ID No.
  • Seq ID No. 145 and Seq ID No. 146 are both hypomethylated probes that target the target region shown in Seq ID No. 17, and Seq ID No. 302 and Seq ID No.
  • Seq ID No. 147 is a hypomethylated probe targeting the target region shown in Seq ID No. 18, and Seq ID No. 304 is a hypermethylated probe targeting the target region shown in Seq ID No. 18.
  • Seq ID No. 148 and Seq ID No. 149 are both hypomethylated probes that target the target region shown in Seq ID No. 19, and Seq ID No. 305 and Seq ID No. 306 are both targeted to Seq ID No.
  • Seq ID No. 150 is a hypomethylated probe targeting the target region shown in Seq ID No. 20, and Seq ID No.
  • Seq ID No. 307 is a hypermethylated probe targeting the target region shown in Seq ID No. 20.
  • Seq ID No. 151 is a hypomethylated probe targeting the target region shown in Seq ID No. 21, and Seq ID No. 308 is a hypermethylated probe targeting the target region shown in Seq ID No. 21.
  • Seq ID No. 152 is a hypomethylated probe targeting the target region shown in Seq ID No. 22, and Seq ID No. 309 is a hypermethylated probe targeting the target region shown in Seq ID No. 22.
  • Seq ID No. 153, Seq ID No. 154, and Seq ID No. 155 are all hypomethylated probes that target the target region shown in Seq ID No. 23, Seq ID No. 310, Seq ID No.
  • Seq ID No. 312 is a hypermethylated probe that targets the target region shown in Seq ID No. 23.
  • Seq ID No. 156 and Seq ID No. 157 are hypomethylated probes that target the target region shown in Seq ID No. 24, and Seq ID No. 313 and Seq ID No. 314 are both targeted to Seq ID No.
  • Seq ID No. 158 and Seq ID No. 159 are both hypomethylated probes that target the target region shown in Seq ID No. 25, and Seq ID No. 315 and Seq ID No. 316 are both targeted to Seq ID No.
  • Seq ID No. 161 are hypomethylation probes that target the target region shown in Seq ID No. 26, and Seq ID No. 317 and Seq ID No. 318 are both targeted to Seq ID No.
  • Seq ID No.162 is a hypomethylated probe targeting the target region shown in Seq ID No.27
  • Seq ID No.319 is a hypermethylated probe targeting the target region shown in Seq ID No.27.
  • Seq ID No. 163 and Seq ID No. 164 are hypomethylation probes that target the target region shown in Seq ID No. 28, and Seq ID No. 320 and Seq ID No. 321 are both targeted to Seq ID No.
  • the hypermethylated probe in the target region shown in .28. Seq ID No. 165 and Seq ID No. 166 are both hypomethylated probes that target the target region shown in Seq ID No. 29, and Seq ID No. 322 and Seq ID No. 323 are both targeted to Seq ID No.
  • Seq ID No. 167 is a hypomethylated probe that targets the target region shown in Seq ID No. 30, and Seq ID No. 324 is a hypermethylated probe that targets the target region shown in Seq ID No. 30.
  • Seq ID No. 168 and Seq ID No. 169 are both hypomethylated probes that target the target region shown in Seq ID No. 31, and Seq ID No.
  • Seq ID No. 325 and Seq ID No. 326 are both targeted to Seq ID No.
  • Seq ID No. 170 is a hypomethylated probe targeting the target region shown in Seq ID No. 32
  • Seq ID No. 327 is a hypermethylated probe targeting the target region shown in Seq ID No. 32.
  • Seq ID No. 171 and Seq ID No. 172 are both hypomethylated probes that target the target region shown in Seq ID No. 33
  • Seq ID No. 328 and Seq ID No. 329 are both targeted to Seq ID No.
  • Seq ID No. 173 is a hypomethylated probe targeting the target region shown in Seq ID No.
  • Seq ID No. 34 is a hypermethylated probe targeting the target region shown in Seq ID No. 34.
  • Seq ID No. 174 and Seq ID No. 175 are hypomethylated probes that target the target region shown in Seq ID No. 35, and Seq ID No. 331 and Seq ID No. 332 are both targeted to Seq ID No.
  • Seq ID No. 176 is a hypomethylated probe targeting the target region shown in Seq ID No. 36
  • Seq ID No. 333 is a hypermethylated probe targeting the target region shown in Seq ID No. 36.
  • Seq ID No. 178 are hypomethylated probes that target the target region shown in Seq ID No. 37, and Seq ID No. 334 and Seq ID No. 335 are both targeted to Seq ID No.
  • Seq ID No. 179 and Seq ID No. 180 are hypomethylated probes that target the target region shown in Seq ID No. 38, and Seq ID No. 336 and Seq ID No. 337 are both targeted to Seq ID No.
  • Seq ID No. 181 is a hypomethylated probe that targets the target region shown in Seq ID No. 39
  • Seq ID No. 338 is a hypermethylated probe that targets the target region shown in Seq ID No.
  • Seq ID No. 182 and Seq ID No. 183 are hypomethylated probes that target the target region shown in Seq ID No. 40, and Seq ID No. 339 and Seq ID No. 340 are both targeted to Seq ID No.
  • Seq ID No. 184 is a hypomethylated probe targeting the target region shown in Seq ID No. 41
  • Seq ID No. 341 is a hypermethylated probe targeting the target region shown in Seq ID No. 41.
  • Seq ID No.185 is a hypomethylated probe targeting the target region shown in Seq ID No.42
  • Seq ID No.342 is a hypermethylated probe targeting the target region shown in Seq ID No.42.
  • Seq ID No. 186 is a hypomethylated probe targeting the target region shown in Seq ID No. 43
  • Seq ID No. 343 is a hypermethylated probe targeting the target region shown in Seq ID No. 43.
  • Seq ID No. 187 is a hypomethylated probe targeting the target region shown in Seq ID No. 44
  • Seq ID No. 344 is a hypermethylated probe targeting the target region shown in Seq ID No. 44.
  • Seq ID No. 188 and Seq ID No. 189 are both hypomethylated probes that target the target region shown in Seq ID No. 45
  • Seq ID No. 345 and Seq ID No. 346 are both targeted to Seq ID No.
  • Seq ID No. 191 is a hypomethylated probe targeting the target region shown in Seq ID No. 47
  • Seq ID No. 348 is a hypermethylated probe targeting the target region shown in Seq ID No. 47.
  • Seq ID No. 192 is a hypomethylated probe targeting the target region shown in Seq ID No. 48
  • Seq ID No. 349 is a hypermethylated probe targeting the target region shown in Seq ID No. 48.
  • Seq ID No. 193 is a hypomethylated probe targeting the target region shown in Seq ID No.
  • Seq ID No. 49, and Seq ID No. 350 is a hypermethylated probe targeting the target region shown in Seq ID No. 49.
  • Seq ID No. 194 is a hypomethylated probe targeting the target region shown in Seq ID No. 50
  • Seq ID No. 351 is a hypermethylated probe targeting the target region shown in Seq ID No. 50.
  • Seq ID No. 195 and Seq ID No. 196 are hypomethylation probes that target the target region shown in Seq ID No. 51, and Seq ID No. 352 and Seq ID No. 353 are both targeted to Seq ID No.
  • Seq ID No.197 is a hypomethylated probe targeting the target region shown in Seq ID No.52
  • Seq ID No.354 is a hypermethylated probe targeting the target region shown in Seq ID No.52.
  • Seq ID No. 198 and Seq ID No. 199 are hypomethylated probes that target the target region shown in Seq ID No. 53, and Seq ID No. 355 and Seq ID No. 356 are both targeted to Seq ID No.
  • Seq ID No. 200 is a hypomethylated probe targeting the target region shown in Seq ID No. 54
  • Seq ID No. 357 is a hypermethylated probe targeting the target region shown in Seq ID No. 54.
  • Seq ID No. 201 and Seq ID No. 202 are hypomethylated probes that target the target region shown in Seq ID No. 55, and Seq ID No. 358 and Seq ID No. 359 are both targeted to Seq ID No.
  • Seq ID No. 203 and Seq ID No. 204 are hypomethylated probes that target the target region shown in Seq ID No. 56, and Seq ID No. 360 and Seq ID No. 361 are both targeted to Seq ID No.
  • Seq ID No. 205 and Seq ID No. 206 are both hypomethylated probes that target the target region shown in Seq ID No. 57, and Seq ID No.
  • Seq ID No. 362 and Seq ID No. 363 are both targeted to Seq ID No.
  • Seq ID No. 207 and Seq ID No. 208 are hypomethylated probes that target the target region shown in Seq ID No. 58, and Seq ID No. 364 and Seq ID No. 365 are both targeted to Seq ID No.
  • Seq ID No. 209 and Seq ID No. 210 are hypomethylated probes that target the target region shown in Seq ID No. 59, and Seq ID No. 366 and Seq ID No. 367 are both targeted to Seq ID No.
  • Seq ID No. 211 is a hypomethylated probe targeting the target region shown in Seq ID No. 60
  • Seq ID No. 368 is a hypermethylated probe targeting the target region shown in Seq ID No. 60.
  • Seq ID No. 212 and Seq ID No. 213 are hypomethylated probes that target the target region shown in Seq ID No. 61, and Seq ID No. 369 and Seq ID No. 370 are both targeted to Seq ID No.
  • Seq ID No. 214 is a hypomethylated probe targeting the target region shown in Seq ID No. 62
  • Seq ID No. 371 is a hypermethylated probe targeting the target region shown in Seq ID No. 62.
  • Seq ID No. 215 is a hypomethylated probe targeting the target region shown in Seq ID No. 63
  • Seq ID No. 372 is a hypermethylated probe targeting the target region shown in Seq ID No. 63
  • Seq ID No.216 is a hypomethylated probe targeting the target region shown in Seq ID No.64
  • Seq ID No.373 is a hypermethylated probe targeting the target region shown in Seq ID No.64
  • Seq ID No. 217 is a hypomethylated probe targeting the target region shown in Seq ID No. 65
  • Seq ID No. 374 is a hypermethylated probe targeting the target region shown in Seq ID No. 65
  • Seq ID No. 218 is a hypomethylated probe targeting the target region shown in Seq ID No.
  • Seq ID No. 66 is a hypermethylated probe targeting the target region shown in Seq ID No. 66.
  • Seq ID No. 219 is a hypomethylated probe targeting the target region shown in Seq ID No. 67
  • Seq ID No. 376 is a hypermethylated probe targeting the target region shown in Seq ID No. 67.
  • Seq ID No. 220 is a hypomethylated probe targeting the target region shown in Seq ID No. 68
  • Seq ID No. 377 is a hypermethylated probe targeting the target region shown in Seq ID No. 68.
  • Seq ID No. 221 is a hypomethylated probe that targets the target region shown in Seq ID No. 69, and Seq ID No.
  • Seq ID No. 378 is a hypermethylated probe that targets the target region shown in Seq ID No. 69.
  • Seq ID No. 222 is a hypomethylated probe targeting the target region shown in Seq ID No. 70
  • Seq ID No. 379 is a hypermethylated probe targeting the target region shown in Seq ID No. 70.
  • Seq ID No. 223 is a hypomethylated probe targeting the target region shown in Seq ID No. 71
  • Seq ID No. 380 is a hypermethylated probe targeting the target region shown in Seq ID No. 71.
  • Seq ID No. 224 is a hypomethylated probe targeting the target region shown in Seq ID No. 72
  • Seq ID No. 381 is a hypermethylated probe targeting the target region shown in Seq ID No. 72.
  • Seq ID No. 225 is a hypomethylated probe targeting the target region shown in Seq ID No. 73
  • Seq ID No. 382 is a hypermethylated probe targeting the target region shown in Seq ID No. 73.
  • Seq ID No. 226 is a hypomethylated probe targeting the target region shown in Seq ID No. 74
  • Seq ID No. 383 is a hypermethylated probe targeting the target region shown in Seq ID No. 74.
  • Seq ID No. 227 is a hypomethylated probe targeting the target region shown in Seq ID No. 75
  • Seq ID No. 384 is a hypermethylated probe targeting the target region shown in Seq ID No. 75.
  • Seq ID No. 229 are both hypomethylated probes that target the target region shown in Seq ID No. 76, and Seq ID No. 385 and Seq ID No. 386 are both targeted to Seq ID No. A hypermethylated probe in the target region shown in .76.
  • Seq ID No. 230 is a hypomethylated probe that targets the target region shown in Seq ID No. 77, and Seq ID No. 387 is a hypermethylated probe that targets the target region shown in Seq ID No. 77.
  • Seq ID No. 231 is a hypomethylated probe targeting the target region shown in Seq ID No. 78, and Seq ID No. 388 is a hypermethylated probe targeting the target region shown in Seq ID No. 78.
  • Seq ID No. 232 is a hypomethylated probe that targets the target region shown in Seq ID No. 79
  • Seq ID No. 389 is a hypermethylated probe that targets the target region shown in Seq ID No. 79.
  • Seq ID No. 233 is a hypomethylated probe targeting the target region shown in Seq ID No. 80
  • Seq ID No. 390 is a hypermethylated probe targeting the target region shown in Seq ID No. 80.
  • Seq ID No. 234 is a hypomethylated probe targeting the target region shown in Seq ID No. 81
  • Seq ID No. 391 is a hypermethylated probe targeting the target region shown in Seq ID No. 81.
  • Seq ID No. 235 is a hypomethylated probe that targets the target region shown in Seq ID No.
  • Seq ID No. 236 is a hypomethylated probe targeting the target region shown in Seq ID No. 83
  • Seq ID No. 393 is a hypermethylated probe targeting the target region shown in Seq ID No. 83.
  • Seq ID No. 237 is a hypomethylated probe targeting the target region shown in Seq ID No. 84
  • Seq ID No. 394 is a hypermethylated probe targeting the target region shown in Seq ID No. 84.
  • Seq ID No. 238 is a hypomethylated probe targeting the target region shown in Seq ID No. 85, and Seq ID No.
  • Seq ID No. 395 is a hypermethylated probe targeting the target region shown in Seq ID No. 85.
  • Seq ID No. 239 is a hypomethylated probe targeting the target region shown in Seq ID No. 86
  • Seq ID No. 396 is a hypermethylated probe targeting the target region shown in Seq ID No. 86.
  • Seq ID No. 240 is a hypomethylated probe that targets the target region shown in Seq ID No. 87
  • Seq ID No. 397 is a hypermethylated probe that targets the target region shown in Seq ID No. 87.
  • Seq ID No. 241 is a hypomethylated probe targeting the target region shown in Seq ID No. 88
  • Seq ID No. 398 is a hypermethylated probe targeting the target region shown in Seq ID No.
  • Seq ID No. 242 is a hypomethylated probe targeting the target region shown in Seq ID No. 89
  • Seq ID No. 399 is a hypermethylated probe targeting the target region shown in Seq ID No. 89.
  • Seq ID No. 243 is a hypomethylated probe that targets the target region shown in Seq ID No. 90
  • Seq ID No. 400 is a hypermethylated probe that targets the target region shown in Seq ID No. 90.
  • Seq ID No. 244 is a hypomethylated probe targeting the target region shown in Seq ID No. 91
  • Seq ID No. 401 is a hypermethylated probe targeting the target region shown in Seq ID No. 91.
  • Seq ID No. 245 is a hypomethylated probe targeting the target region shown in Seq ID No. 92
  • Seq ID No. 402 is a hypermethylated probe targeting the target region shown in Seq ID No. 92
  • Seq ID No. 246 is a hypomethylated probe targeting the target region shown in Seq ID No. 93
  • Seq ID No. 403 is a hypermethylated probe targeting the target region shown in Seq ID No. 93
  • Seq ID No. 247 is a hypomethylated probe targeting the target region shown in Seq ID No. 94
  • Seq ID No. 404 is a hypermethylated probe targeting the target region shown in Seq ID No. 94
  • Seq ID No. 248 is a hypomethylated probe targeting the target region shown in Seq ID No.
  • Seq ID No. 405 is a hypermethylated probe targeting the target region shown in Seq ID No. 95.
  • Seq ID No. 249 is a hypomethylated probe that targets the target region shown in Seq ID No. 96
  • Seq ID No. 406 is a hypermethylated probe that targets the target region shown in Seq ID No. 96.
  • Seq ID No. 250 is a hypomethylated probe targeting the target region shown in Seq ID No. 97
  • Seq ID No. 407 is a hypermethylated probe targeting the target region shown in Seq ID No. 97.
  • Seq ID No. 251 is a hypomethylated probe targeting the target region shown in Seq ID No. 98, and Seq ID No.
  • Seq ID No. 408 is a hypermethylated probe targeting the target region shown in Seq ID No. 98.
  • Seq ID No. 252 is a hypomethylated probe that targets the target region shown in Seq ID No. 99
  • Seq ID No. 409 is a hypermethylated probe that targets the target region shown in Seq ID No. 99.
  • Seq ID No.253 is a hypomethylated probe targeting the target region shown in Seq ID No.100
  • Seq ID No.410 is a hypermethylated probe targeting the target region shown in Seq ID No.100.
  • Seq ID No. 254 is a hypomethylated probe targeting the target region shown in Seq ID No. 101
  • Seq ID No. 411 is a hypermethylated probe targeting the target region shown in Seq ID No. 101.
  • Seq ID No. 255 and Seq ID No. 256 are both hypomethylated probes that target the target region shown in Seq ID No. 102, and Seq ID No. 412 and Seq ID No. 413 are both targeted to Seq ID No.
  • Seq ID No. 257 is a hypomethylated probe targeting the target region shown in Seq ID No. 103, and Seq ID No. 414 is a hypermethylated probe targeting the target region shown in Seq ID No. 103.
  • Seq ID No. 258 is a hypomethylated probe targeting the target region shown in Seq ID No. 104, and Seq ID No. 415 is a hypermethylated probe targeting the target region shown in Seq ID No. 104.
  • Seq ID No. 259 is a hypomethylated probe targeting the target region shown in Seq ID No. 105
  • Seq ID No. 416 is a hypermethylated probe targeting the target region shown in Seq ID No. 105
  • Seq ID No. 260 is a hypomethylated probe targeting the target region shown in Seq ID No. 106
  • Seq ID No. 417 is a hypermethylated probe targeting the target region shown in Seq ID No. 106
  • Seq ID No. 261 is a hypomethylated probe targeting the target region shown in Seq ID No. 107
  • Seq ID No. 418 is a hypermethylated probe targeting the target region shown in Seq ID No. 107.
  • Seq ID No. 262 is a hypomethylated probe targeting the target region shown in Seq ID No. 108
  • Seq ID No. 419 is a hypermethylated probe targeting the target region shown in Seq ID No. 108.
  • Seq ID No. 263 is a hypomethylated probe targeting the target region shown in Seq ID No. 109
  • Seq ID No. 420 is a hypermethylated probe targeting the target region shown in Seq ID No. 109.
  • Seq ID No.264, Seq ID No.265 and Seq ID No.266 are all hypomethylated probes that target the target region shown in Seq ID No.110, Seq ID No.421, Seq ID No.422 and Seq ID No.
  • Seq ID No. 423 are all hypermethylated probes that target the target region shown in Seq ID No. 110.
  • Seq ID No. 267 is a hypomethylated probe targeting the target region shown in Seq ID No. 111
  • Seq ID No. 424 is a hypermethylated probe targeting the target region shown in Seq ID No. 111.
  • Seq ID No. 268 is a hypomethylated probe targeting the target region shown in Seq ID No. 112
  • Seq ID No. 425 is a hypermethylated probe targeting the target region shown in Seq ID No. 112.
  • Seq ID No. 269 is a hypomethylated probe targeting the target region shown in Seq ID No. 113
  • Seq ID No. 426 is a hypermethylated probe targeting the target region shown in Seq ID No. 113.
  • Seq ID No. 270 and Seq ID No. 271 are both hypomethylated probes that target the target region shown in Seq ID No. 114, and Seq ID No. 427 and Seq ID No. 428 are both targeted to Seq ID No.
  • Seq ID No. 272 is a hypomethylated probe targeting the target region shown in Seq ID No. 115
  • Seq ID No. 429 is a hypermethylated probe targeting the target region shown in Seq ID No. 115.
  • Seq ID No. 273 is a hypomethylated probe targeting the target region shown in Seq ID No. 116
  • Seq ID No. 430 is a hypermethylated probe targeting the target region shown in Seq ID No. 116.
  • Seq ID No.274 is a hypomethylated probe targeting the target region shown in Seq ID No.117
  • Seq ID No.431 is a hypermethylated probe targeting the target region shown in Seq ID No.117.
  • Table 1 shows the target sequence targeted by the probe.
  • the length of each probe in the above-mentioned probe composition is 40-60 bp, preferably 45-56 bp, preferably 50-56 bp, and more preferably 50 bp.
  • the application also provides a kit.
  • the kit includes the probe composition described in any of the above embodiments.
  • the application also provides the use of the probe composition.
  • the above-mentioned probe composition is used to prepare and detect esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and intrauterine cancer.
  • Membrane cancer kit is used to prepare and detect esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and intrauterine cancer.
  • the application also provides a chip.
  • the probe composition described in any of the above embodiments is immobilized on the chip.
  • the application also provides a method for detecting 11 kinds of esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer by using the probe composition Methods of cancer.
  • the detection method uses hybrid capture to enrich cfDNA, and uses NGS technology to detect methylation sites that are highly related to cancer. It covers the six malignant tumors with the highest incidence in my country (lung cancer, gastric cancer, colorectal cancer, liver cancer, breast cancer and esophageal cancer), as well as pancreatic cancer, which is extremely malignant. It also includes three other female tumors and one male tumor (cervical cancer, ovarian cancer, endometrial cancer, and prostate cancer). Finally, based on the detection of gene methylation changes in plasma cfDNA, it provides information for early screening and early diagnosis of multiple cancers.
  • This application also provides a method for simultaneously detecting changes in the methylation level of the 11 cancers mentioned above by using the kit.
  • this application relates to 11 in vitro cancer detection methods for subjects, including the following steps: collecting a sample of the subject; extracting and purifying the DNA in the sample; constructing a DNA library for sequencing based on the purified DNA sample; Bisulfite transforms the constructed DNA library; pre-PCR amplifies the bisulfite-converted DNA library; uses the probe composition to hybridize and capture the sample amplified by pre-PCR; Products captured by hybridization; perform high-throughput next-generation sequencing on the products amplified by PCR and captured by hybridization; analyze the sequencing data to determine the methylation level of the sample; judge based on the methylation level of the sample The condition of the patient.
  • the subject is suspected of having cancer.
  • the sample collected from the subject is a plasma sample.
  • the conversion is treatment with bisulfite.
  • the probe composition includes: 2 probes targeting a pan-cancer specific region, n probes targeting a cancer-specific region, and m probes targeting a tissue-specific region.
  • the probe composition includes: a hypomethylation probe that hybridizes to the cancer-specific region, pan-cancer specific region, and tissue-specific region that are converted by bisulfite and do not contain CG methylation , And a hypermethylation probe that hybridizes to the cancer-specific region, pan-cancer-specific region, and tissue-specific region where the bisulfite-converted CG is fully methylated.
  • the length of each probe in the probe composition is 40-60 bp.
  • the length of each probe in the probe composition is 45-56 bp, preferably 50-56 bp, and more preferably 50 bp.
  • the n probes in the probe composition target a cancer-specific region, where n is any integer selected from 1-192; wherein, the cancer-specific region is selected from Seq ID No.: Any of 1-62.
  • the m probes in the probe composition target the tissue-specific region, where m is an integer selected from 1-116; wherein, the tissue-specific region is selected from Seq ID No. : Any of 65-117.
  • the hypomethylation probes include probes targeting cancer-specific regions Seq ID No.: any of 118-214, and probes targeting pan-cancer specific regions Seq ID No.: 215-216 Any, and the probe Seq ID No. that targets tissue-specific regions: Any of 217-274.
  • the hypermethylation probes include probes targeting cancer-specific regions, Seq ID No.: any of 275-371, and probes targeting pan-cancer specific regions, Seq ID No.: 372-373, Seq ID No.: any of 374-431 probes targeting tissue-specific regions.
  • the interpretation includes the following steps: (1) compare the pan-cancer specific region database and perform interpretation to confirm whether the subject has cancer; (2) compare the cancer specific region database and perform interpretation To confirm that the subject's cancer is one of several suspected cancers; (3) Compare the tissue-specific area database and perform interpretation to confirm the subject's cancer site.
  • the step (1) includes the following interpretation: judging whether the methylation level of the pan-cancer specific region Seq ID No.: 63 is greater than or equal to 55%, and the pan-cancer specific region Seq ID No.: 64 If the methylation level of is greater than or equal to 60%, it is judged that the patient has cancer.
  • the step (2) includes the following interpretation: if among the n probes targeting the cancer-specific region, the methylation level of the region targeted by n1 probes is greater than or equal to the respective threshold, and n1/n ⁇ 20%, preferably n1/n ⁇ 30%, then it is judged that the patient has any one of the tissue-specific cancers.
  • the step (3) includes the following interpretation: among the m probes targeting the tissue-specific region, the methylation level of the region targeted by m1 probes is greater than or equal to the respective threshold, and then further analysis is greater than or equal to the respective threshold.
  • Threshold m1 probes target tissues and count the number of probes in each tissue that are greater than or equal to the threshold, and judge that the tissue in which the patient suffers from cancer is the tissue with the largest number of probes with methylation level greater than or equal to the threshold. Table 1 below also shows the methylation threshold of each target region.
  • Agilent 2100 performs fragment detection, and Qubit is directly used in subsequent experiments.
  • DNA protection buffer added to the liquid turns blue. Mix gently by pipetting, and then divide into two tubes on the PCR machine.
  • the number of PCR cycles is adjusted according to the amount of DNA input.
  • the reference data is as follows:
  • the length of the library is about 270bp-320bp.
  • Hyb buffer solution at room temperature to melt, there will be precipitation after melting, and place it in a 65°C water bath to preheat it after mixing. After completely dissolved (no precipitation and turbidity), take 20 ⁇ l Hyb buffer solution and place In the new 200 ⁇ l PCR tube, close the tube cap, mark it as A, and continue to incubate it in a 65°C water bath for later use.
  • Seq ID No. of probes targeting cancer-specific regions any of 275-371
  • Methylation bio-information analysis process It is roughly as follows: Use quality control software such as trimmomatic to check the quality of sequencing, remove low-quality reads, and then use comparison software such as Bismarker to compare the clean data after quality control to the reference genome, and use R packages such as methykit to extract the corresponding Methylation site. Finally, calculate the methylation ratio of each target area on the Panel.
  • quality control software such as trimmomatic to check the quality of sequencing, remove low-quality reads
  • comparison software such as Bismarker to compare the clean data after quality control to the reference genome
  • R packages such as methykit
  • pan-cancer specific markers TBX15 and CRYGD genes greater than or equal to 55% and 60%, and then preliminarily determine that the sample is a sample suffering from cancer;
  • the methylation levels of cancer-specific markers OTX1, SFRP2, CDO1, TRIM15, ALX4, and CCNA1 are greater than or equal to the respective thresholds shown in Table 1 (as shown in Table 19 above), and then the sample is further judged as suffering from the following 11 types
  • a sample of any cancer esophageal cancer, stomach cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer
  • interpret tissue-specific markers Based on the target regions that are greater than or equal to their respective thresholds in Table 19, it can be seen that there are 6 target regions specific to gastric tissue Seq ID No.
  • Seq ID No. 82, Seq ID No. 97, Seq ID No. 98, Seq ID No. 99 and Seq ID No. 100 are greater than or equal to their respective thresholds of methylation levels, and no other tissue-specific markers have methylation greater than or equal to their respective thresholds, so they are greater than or equal to their respective methylation thresholds Among the tissue-specific markers, gastric tissue-specific markers are the most, and the sample is finally judged to be a sample with gastric cancer.
  • the patient’s blood was drawn again 48 hours after the operation, and the peripheral blood was collected using the Panel test of the application according to the method of Example 1.
  • the database was constructed and sequenced on the Illumina platform; the sequencing data was subjected to the above-mentioned biological information analysis process and passed pattern recognition By analyzing all the sequencing data, the results show that the gene methylation level in the above table has returned to the normal level.
  • pan-cancer specific markers TBX15 and CRYGD genes greater than or equal to 55% and 60%, and then preliminarily determine that the sample is a sample suffering from cancer;
  • the methylation levels of the cancer-specific markers TRH, CDO1, ELMO1, GFRA1, CCNA1, and SALL1 are greater than or equal to the respective thresholds shown in Table 1 (as shown in Table 20 above), and then the sample is further judged as suffering from the following 11 types
  • a sample of any cancer esophageal cancer, stomach cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer
  • interpret tissue-specific markers Based on the target regions in Table 20 that are greater than or equal to their respective thresholds, it can be seen that there are 6 target regions specific to colorectal tissues Seq ID No.
  • Seq ID No. 64 Seq ID No. 87, Seq ID No. 88 , Seq ID No. 89, Seq ID No. 90 are greater than or equal to their respective thresholds of methylation levels, and no other tissue-specific markers have methylation greater than or equal to their respective thresholds, so they are greater than or equal to their respective methylation thresholds.
  • threshold tissue-specific markers colorectal tissue-specific markers are the most, and the sample is finally judged to be a sample with colorectal cancer.
  • a lung cancer sample was detected by the Panel of this application, and peripheral blood was collected according to the method in Example 1; the database was constructed and sequenced on the Illumina platform; the sequencing data was subjected to the above-mentioned biological information analysis process to obtain the methylation level.
  • the results are shown in Table 21 As shown (Table 21 shows the detected target regions greater than or equal to the methylation threshold).
  • pan-cancer specific markers TBX15 and CRYGD genes greater than or equal to 55% and 60%, and then preliminarily determine that the sample is a sample suffering from cancer;
  • the methylation levels of cancer-specific markers RUNX3, APC, HOXA11AS, PHOX2A, and GALR1 are greater than or equal to the respective thresholds shown in Table 1 (as shown in Table 21 above), and the sample is further judged to have the following 11 cancers ( Esophageal cancer, stomach cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and endometrial cancer) samples of any cancer; finally, the interpretation of tissue-specific markers, based on In Table 21, the target regions that are greater than or equal to their respective thresholds can be seen as five target regions Seq ID No.
  • Seq ID No. 72 Seq ID No. 76, Seq ID No. 91, and Seq ID that are specific to lung tissue No. 92 is greater than or equal to the respective threshold of methylation level, and no other tissue-specific markers have methylation greater than or equal to their respective thresholds. Therefore, in the tissue-specific markers greater than or equal to their respective methylation thresholds, If the lung tissue has the most specific markers, the sample is finally judged to be a sample with lung cancer.
  • a liver cancer sample was detected by the Panel of this application, and peripheral blood was collected according to the method of Example 1; the database was constructed and sequenced on the Illumina platform; the sequencing data was subjected to the above-mentioned biological information analysis process to obtain the methylation level.
  • the results are shown in Table 22 As shown (Table 22 shows the detected target regions greater than or equal to the methylation threshold).
  • Seq ID No. 74 that are specific to liver tissue are greater than or equal to their respective thresholds of methylation levels, and there are no other tissues
  • the methylation of specific markers is greater than or equal to their respective thresholds. Therefore, among the tissue-specific markers greater than or equal to their respective methylation thresholds, liver tissue-specific markers are the most, and the sample is finally judged to be a sample with liver cancer .
  • a sample of esophageal cancer was detected by the Panel of this application, and peripheral blood was collected according to the method of Example 1; the database was built and sequenced on the Illumina platform; the sequencing data was subjected to the above-mentioned biological information analysis process to obtain the methylation level.
  • the results are as follows 23 (Table 23 shows the detected target regions greater than or equal to the methylation threshold).
  • Seq ID No. 67 Seq ID No. 79, and Seq ID No. 80 are greater than or equal to each The threshold of methylation level, no other tissue-specific markers have methylation greater than or equal to their respective thresholds. Therefore, among the tissue-specific markers greater than or equal to their respective methylation thresholds, esophageal tissue-specific markers At most, the sample is finally judged to be a sample suffering from esophageal cancer.
  • pancreatic cancer was detected by the Panel of this application, and peripheral blood was collected according to the method in Example 1; the database was constructed and sequenced on the Illumina platform; the sequencing data was subjected to the above-mentioned biological information analysis process to obtain the methylation level.
  • the results are as follows 24 (Table 24 shows the detected target regions greater than or equal to the methylation threshold).
  • pan-cancer specific markers TBX15 and CRYGD genes greater than or equal to 55% and 60%, and then preliminarily determine that the sample is a sample suffering from cancer;
  • the methylation levels of the cancer-specific markers HOPX, SFRP2, GFRA1, HOXB4, and SALL1 are greater than or equal to the respective thresholds shown in Table 1 (as shown in Table 24 above), and the sample is further judged to have the following 11 cancers ( Esophageal cancer, stomach cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and endometrial cancer) samples of any cancer; finally, the interpretation of tissue-specific markers, based on In Table 24, the target regions that are greater than or equal to their respective thresholds can be seen as five target regions Seq ID No.
  • Seq ID No. 112 Seq ID No. 113, Seq ID No. 114, and Seq ID that are specific to pancreatic tissue No. 115 is greater than or equal to the respective threshold of methylation level, and no other tissue-specific markers have methylation greater than or equal to their respective thresholds. Therefore, among tissue-specific markers greater than or equal to their respective methylation thresholds, The pancreatic tissue-specific markers are the most, and the sample is finally judged to be a sample with pancreatic cancer.
  • a breast cancer sample was detected by the Panel of this application, and peripheral blood was collected according to the method of Example 1; the database was constructed and sequenced on the Illumina platform; the sequencing data was subjected to the above-mentioned biological information analysis process to obtain the methylation level.
  • the results are as follows 25 (Table 25 shows the detected target regions greater than or equal to the methylation threshold).
  • Seq ID No. 85, and Seq ID No. 86 are greater than or equal to their respective methylation.
  • No other tissue-specific markers have methylation greater than or equal to their respective thresholds. Therefore, among the tissue-specific markers greater than or equal to their respective methylation thresholds, breast tissue-specific markers are the most, and the final The sample is judged to be a sample suffering from breast cancer.

Abstract

Disclosed is a probe composition. By means of the meta-analysis of methylation data of a TCGA database and a GEO database, the composition screens 6.5 Kbp of a capture region, which capture region comprises up to 66 methylation change genes and non-coding DNA regions highly related to cancers. The method can be used to detect methylation level changes of 11 common cancers at once: esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, etc. The method can be used for the early screening of an asymptomatic population and the prognostic detection of cancer patients.

Description

一种检测11种癌症的探针组合物A probe composition for detecting 11 kinds of cancer 技术领域Technical field
本申请涉及一种癌症基因甲基化检测组合物，特别涉及一种特异性识别重亚硫酸盐(Bisulfite)处理后的DNA序列的探针组合物，以及基于高通量测序(NGS)方法检测食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌等11种肿瘤游离DNA甲基化水平变化的应用。This application relates to a cancer gene methylation detection composition, in particular to a probe composition that specifically recognizes the DNA sequence after Bisulfite treatment, and detection based on high-throughput sequencing (NGS) method Application of changes in the free DNA methylation level of 11 tumors including esophageal cancer, stomach cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and endometrial cancer.
背景技术Background technique
高通量测序(NGS)技术是现代基因组学研究领域革命性的创新，该技术可以同时对几十到几百万条DNA分子进行序列分析，这标志着后基因组时代的到来。通过对测序深度的控制，可实现从头测序和重测序等不同的目标，也可以通过不同的前期处理，分析基因组、转录组和甲基化组的序列。High-throughput sequencing (NGS) technology is a revolutionary innovation in the field of modern genomics research. This technology can simultaneously sequence dozens to millions of DNA molecules, marking the arrival of the post-genome era. Through the control of the sequencing depth, different goals such as de novo sequencing and resequencing can be achieved, and the sequence of the genome, transcriptome, and methylome can also be analyzed through different pre-processing.
目前临床基因检测的技术主要是聚合酶链式反应(PCR)、荧光原位杂交(FISH)和基因芯片技术。PCR仪器设备价格低，灵敏性高，操作简单快速，在临床的普及度高，但是受技术限制，只能同时检测少数几个基因。FISH灵敏度高，但操作难度较大。基因芯片通量高于前两者，可以同时检测大量的基因。但局限在于只能检测已知的基因或变异，准确性低，假阳性高。而NGS技术具有通量高(同时检测大量已知和未知的基因及变异)，结果准确(准确率高于基因芯片)，检测速度较快，均摊到每个基因的检测成本低等特点，现在已经逐渐应用到临床疾病检测和监控等领域。随着未来测序费用的进一步降低，NGS必然会逐步取代基因芯片等其他高通量技术。The current clinical gene detection technologies are mainly polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH) and gene chip technology. PCR equipment has low price, high sensitivity, simple and fast operation, and high clinical popularity. However, due to technical limitations, it can only detect a few genes at the same time. FISH is highly sensitive but difficult to operate. The gene chip throughput is higher than the former two, and it can detect a large number of genes at the same time. But the limitation is that it can only detect known genes or mutations, with low accuracy and high false positives. The NGS technology has the characteristics of high throughput (detecting a large number of known and unknown genes and mutations at the same time), accurate results (higher accuracy than gene chips), fast detection speed, and low cost of detection for each gene. Now Has been gradually applied to clinical disease detection and monitoring and other fields. With the further reduction of sequencing costs in the future, NGS will inevitably gradually replace other high-throughput technologies such as gene chips.
由于目前常规全基因组测序的总价比较高，如果为了检测稀有变异而增加测序深度，则最终价格让消费者难以承受。因此目标序列捕获测序成为比较主流的选择，该技术是根据检测的需求，针对感兴趣的基因组区域设计捕获探针，通过杂交互补的原理富集目标片段DNA，并在后续进行NGS检测。 这种策略可以根据研究或检测的目的进行灵活定制，只选择少量的基因区域，加大测序深度，可以有效的发现目标区域变异的状况，具有很高的灵敏度和准确性。Since the total price of conventional whole-genome sequencing is relatively high, if the depth of sequencing is increased in order to detect rare variants, the final price will be unbearable for consumers. Therefore, target sequence capture and sequencing has become a more mainstream choice. This technology is based on detection requirements, designing capture probes for the genomic region of interest, enriching the target fragment DNA through the principle of hybridization and complementation, and subsequently performing NGS detection. This strategy can be flexibly customized according to the purpose of research or detection, selecting only a small number of gene regions, increasing the depth of sequencing, and effectively discovering the mutation status of the target region, with high sensitivity and accuracy.
在癌症的发生和进展过程中，遗传信息会产生一系列的变化，包括DNA的突变、插入/缺失，染色体结构变异，拷贝数变异，以及表观遗传信息的改变。在癌症的演进过程中，DNA序列的变异是随机发生的，仅当变异发生在关键的生长控制基因时，才可以导致恶性肿瘤的产生。而大多数的基因表达异常是源于表观遗传的改变，通常是DNA甲基化水平的改变。研究表明，基因甲基化水平的改变早于基因变异，跟踪检测基因甲基化的变化，可以较早的预测癌症产生。近年来，随着基因组学的发展，现在已经对超过30种癌症的表观基因组进行了研究。结果显示，尽管DNA甲基化并非在每种癌中都占主导地位，但毫无疑问的是，基因甲基化修饰模式的变化会改变细胞的发展倾向以及肿瘤的表型，从而对大多数癌症的发生发展起重要的影响。During the occurrence and progression of cancer, genetic information will produce a series of changes, including DNA mutations, insertions/deletions, chromosomal structure variations, copy number variations, and changes in epigenetic information. During the evolution of cancer, DNA sequence mutations occur randomly, and only when mutations occur in key growth control genes, can it lead to malignant tumors. Most gene expression abnormalities are due to epigenetic changes, usually changes in DNA methylation levels. Studies have shown that changes in gene methylation levels are earlier than gene mutations. Tracking and detecting changes in gene methylation can predict the occurrence of cancer earlier. In recent years, with the development of genomics, the epigenome of more than 30 cancers has now been studied. The results show that although DNA methylation does not predominate in every cancer, there is no doubt that changes in gene methylation modification patterns will change the developmental tendency of cells and the phenotype of tumors, thereby affecting most cancers. The occurrence and development of cancer have an important impact.
2018年初癌症基因组图谱(TCGA)发表了27篇总结性的分析文章，对历时十多年的30多种癌症数据做了迄今为止最全面的泛癌基因组分析。在通过整合染色体变异、DNA甲基化、RNA和蛋白等多种数据进行分析后，发现，解剖学上的33中癌症可以根据分子特征分成28个亚型。在某种分子亚型会包括25种传统意义上的癌症。这个结果说明，来源于不同器官的癌症存在共同的分子特征。与此同时，来源于同一器官的癌症可能有不同的基因组图谱。由此可见，在不远的将来，癌症筛查和诊断标志物的开发必将引入更多泛癌的概念，不仅研究解剖学层次的癌症，更应在分子水平研究癌症，开发出可以覆盖某种分子分型的泛癌标志物。At the beginning of 2018, the Cancer Genome Atlas (TCGA) published 27 conclusive analysis articles, and made the most comprehensive pan-cancer genome analysis to date on more than 30 kinds of cancer data that lasted for more than ten years. After analyzing various data such as chromosome variation, DNA methylation, RNA and protein, it was found that 33 anatomical cancers can be divided into 28 subtypes based on molecular characteristics. In a certain molecular subtype will include 25 kinds of cancer in the traditional sense. This result indicates that cancers from different organs have common molecular characteristics. At the same time, cancers from the same organ may have different genome profiles. It can be seen that in the near future, the development of cancer screening and diagnostic markers will surely introduce more pan-cancer concepts, not only studying cancer at the anatomical level, but also studying cancer at the molecular level. A molecularly typed pan-cancer marker.
液体活检是体外诊断的一种方式，采用非侵入性的血液检测，可以监测肿瘤或转移灶释放到血液的循环肿瘤细胞(CTC)或循环肿瘤DNA(ctDNA)，该技术可以有效降低侵入性造成的危害，且能够实现对肿瘤各部分和所有转移灶的采样，克服肿瘤异质性(而目前采用的标准组织活检只能反映肿瘤某一部分的特征)，并实现实时监测，具有更高的灵敏度，甚至有可能通过基因组信息预测发生病变的部位，可以有效延长患者生存期。根据这些优点，液体活检可以用于肿瘤的早诊、辅助分期、预后和复发监测，用药指导等方面。目前最常用于的液体活检的游离DNA。Liquid biopsy is a method of in vitro diagnosis. It uses non-invasive blood testing to monitor circulating tumor cells (CTC) or circulating tumor DNA (ctDNA) released into the blood by tumors or metastases. This technology can effectively reduce invasiveness. It can realize the sampling of all parts of the tumor and all metastases, overcome tumor heterogeneity (and the current standard tissue biopsy can only reflect the characteristics of a certain part of the tumor), and realize real-time monitoring with higher sensitivity , It is even possible to predict the location of the lesion based on genomic information, which can effectively prolong the survival time of patients. Based on these advantages, liquid biopsy can be used for early diagnosis of tumors, auxiliary staging, prognosis and recurrence monitoring, medication guidance and other aspects. Currently the most commonly used liquid biopsy of free DNA.
游离DNA(cfDNA)是存在于循环血中游离与细胞外的部分降解的内源性DNA。研究表明，肿瘤组织在发生发展的过程中，其肿瘤细胞凋亡后，会向血浆中释放DNA，经降解后，形成游离肿瘤DNA(ctDNA)。CtDNA的分子遗传特征(如基因突变，微卫星不稳定性和抑癌基因启动子甲基化等)与肿瘤组织DNA相一致。在多癌的早期筛查和检测中，采集外周血比其它临床检测手段更为简便、易于向基层推广，而且由于其无创的特点，更易于被无症状人群接受。因此通过检测血浆中的ctDNA甲基化水平改变，可以成为多癌早期筛查和诊断的重要手段之一。Cell-free DNA (cfDNA) is a partially degraded endogenous DNA that exists in circulating blood and is free and extracellular. Studies have shown that in the process of tumor tissue development, after tumor cell apoptosis, DNA will be released into plasma, and after degradation, free tumor DNA (ctDNA) will be formed. The molecular genetic characteristics of CtDNA (such as gene mutation, microsatellite instability, and tumor suppressor gene promoter methylation, etc.) are consistent with tumor tissue DNA. In the early screening and detection of multiple cancers, the collection of peripheral blood is simpler than other clinical detection methods, easy to promote to the grassroots, and because of its non-invasive characteristics, it is easier to be accepted by asymptomatic people. Therefore, the detection of changes in the level of ctDNA methylation in plasma can become one of the important methods for early screening and diagnosis of multiple cancers.
利用目标序列捕获技术结合NGS监测cfDNA的变异以及甲基化水平变化，可以实现肿瘤早期筛查、易感基因监测、伴随诊断、个性化用药、预后监测等各方面的应用。目前国内外多个公司都推出了不同规模，针对不同应用场景的癌症检测panel，有部分panel已经获得了FDA或CFDA的批准文号。如Foundation Medicine推出的FoundationOne CDx覆盖324个基因，纪念斯隆·凯特琳癌症研究中心(MSK)推出的IMPACT覆盖468种癌症相关基因，燃石医学推出的“人EGFR/ALK/BRAF/KRAS基因突变联合检测试剂盒”，诺禾致源推出的“人EGFR、KRAS、BRAF、PIK3CA、ALK、ROS1基因突变检测试剂盒”等。鹍远基因也于2018年推出了检测结直肠癌甲基化水平的产品“常乐思”。Using target sequence capture technology combined with NGS to monitor cfDNA variation and methylation level changes can realize early tumor screening, susceptibility gene monitoring, companion diagnosis, personalized medication, prognostic monitoring and other applications. At present, many companies at home and abroad have launched cancer detection panels of different scales for different application scenarios. Some panels have obtained FDA or CFDA approval numbers. For example, FoundationOne CDx launched by Foundation Medicine covers 324 genes, IMPACT launched by Memorial Sloan Kettering Cancer Research Center (MSK) covers 468 cancer-related genes, and Burning Rock Medicine launched "human EGFR/ALK/BRAF/KRAS gene mutations" Joint detection kit”, “Human EGFR, KRAS, BRAF, PIK3CA, ALK, ROS1 gene mutation detection kit” launched by Nuohe Zhiyuan, etc. Kunyuan Gene also launched the product "Chang Lesi" to detect the methylation level of colorectal cancer in 2018.
发明内容Summary of the invention
综上所述，针对目前多癌检测产品的缺乏和技术限制，本申请提供了一种探针组合物，可用于食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌等11种癌症的早期筛查。该探针组合物可以：1)以非侵入性的方式用于无症状人群的早期筛查，以及癌症患者的预后检测，降低了侵入性检测造成的危害，2)加大了测序深度，使检测基因的广度优于现有的技术和产品，具有通量高，检测速度较快，均摊到每个基因的检测成本低等特点，3)能够实现对肿瘤各部分和所有转移灶的采样，克服肿瘤异质性，和4)具有更高的灵敏度和准确性，能够实现实时监测，甚至有可能通过基因组信息预测发生病变的部位有效延长患者生存期。In summary, in view of the lack of current multi-cancer detection products and technical limitations, this application provides a probe composition that can be used for esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, and breast cancer. Early screening of 11 types of cancer, including cancer, ovarian cancer, cervical cancer and endometrial cancer. The probe composition can: 1) be used in a non-invasive way for early screening of asymptomatic people and prognostic detection of cancer patients, reducing the harm caused by invasive detection, 2) increasing the depth of sequencing and making The breadth of gene detection is better than existing technologies and products, with high throughput, faster detection speed, and low cost of detection for each gene. 3) It can sample all parts of the tumor and all metastases. Overcome tumor heterogeneity, and 4) have higher sensitivity and accuracy, can realize real-time monitoring, and it is even possible to predict the location of lesions through genomic information to effectively prolong the survival of patients.
具体地，本申请涉及如下内容：Specifically, this application involves the following:
1.一种探针组合物，其包括：靶向食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌中的任意癌症的特异性区域的探针，其中，所述癌症特异性区域选自Seq ID No.：1-62中的任意。1. A probe composition comprising: targeting any cancer among esophageal cancer, stomach cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer The specific region of the probe, wherein the cancer-specific region is selected from any of Seq ID No.: 1-62.
2.根据第1项所述的探针组合物，其还包括：靶向泛癌特异性区域中的任意的探针，所述泛癌特异性区域选自Seq ID No.：63-64中的任意。2. The probe composition according to item 1, further comprising: any probe targeting a pan-cancer specific region, the pan-cancer specific region selected from Seq ID No.: 63-64 Arbitrarily.
3.根据第1项所述的探针组合物，其还包括：靶向组织特异性区域中的任意的探针，所述组织特异性区域选自Seq ID No.：65-117中的任意。3. The probe composition according to item 1, further comprising: any probe that targets a tissue-specific region, the tissue-specific region is selected from any of Seq ID No.: 65-117 .
4.根据第3项所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：66-67、79、81-82、97-102、116-117是食管的组织特异性靶区域。4. The probe composition according to item 3, characterized in that the Seq ID No.: 66-67, 79, 81-82, 97-102, 116-117 in the tissue-specific region is esophageal Tissue specific target area.
5.根据第3项所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：81、82、97-102是胃的组织特异性靶区域。5. The probe composition according to item 3, characterized in that Seq ID Nos.: 81, 82, 97-102 in the tissue-specific region are tissue-specific target regions of the stomach.
6.根据第3项所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：83、87-90、110是结直肠的组织特异性靶区域。6. The probe composition according to item 3, characterized in that Seq ID No. 83, 87-90, 110 in the tissue-specific region are tissue-specific target regions of the colorectal.
7.根据第3项所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：65、70-72、76、91-92、103-104是肺的组织特异性靶区域。7. The probe composition according to item 3, characterized in that Seq ID No. 65, 70-72, 76, 91-92, 103-104 in the tissue-specific region are tissue-specific of the lung Sexual target area.
8.根据第3项所述的探针组合物，其特征在于，所述组织特异性区域中：Seq ID No.：73-74、107是肝的组织特异性靶区域。8. The probe composition according to item 3, characterized in that, among the tissue-specific regions: Seq ID No.: 73-74 and 107 are tissue-specific target regions of the liver.
9.根据第3项所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：111-115是胰腺的组织特异性靶区域。9. The probe composition according to item 3, wherein Seq ID No.: 111-115 in the tissue-specific region is a tissue-specific target region of the pancreas.
10.根据第3项所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：80、84、93、95是前列腺的组织特异性靶区域。10. The probe composition according to item 3, characterized in that Seq ID No. 80, 84, 93, 95 in the tissue-specific region are tissue-specific target regions of the prostate.
11.根据第3项所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：78、84-86是乳腺的组织特异性靶区域。11. The probe composition according to item 3, characterized in that Seq ID Nos.: 78 and 84-86 in the tissue-specific region are tissue-specific target regions of the breast.
12.根据权利要求3所述的探针组合物，其特征在于，在所述组织特异性区域中，Seq ID No.：77、96、105是卵巢的组织特异性靶区域。The probe composition according to claim 3, wherein in the tissue-specific region, Seq ID No.: 77, 96, and 105 are tissue-specific target regions of the ovary.
13.根据第3项所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：68-69、75、109是宫颈的组织特异性靶区域。13. The probe composition according to item 3, characterized in that Seq ID No.: 68-69, 75, 109 in the tissue-specific region are tissue-specific target regions of the cervix.
14.根据第3项所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：94、106、108是子宫内膜的组织特异性靶区域。14. The probe composition according to item 3, characterized in that Seq ID Nos. 94, 106, 108 in the tissue-specific region are tissue-specific target regions of the endometrium.
15.根据第1-14项中任一项所述的探针组合物，其包括：低甲基化探针，其与经重亚硫酸盐转化的不含CG甲基化的所述特异性区域杂交，和；高甲基化探针，其与经重亚硫酸盐转化的CG全部甲基化的所述特异性区域杂交。15. The probe composition according to any one of items 1-14, comprising: a hypomethylation probe, which is compatible with the bisulfite-converted CG-free CG methylation specificity Regional hybridization, and; a hypermethylated probe, which hybridizes to the specific region where the bisulfite-converted CG is fully methylated.
16.根据第15项所述的探针组合物，其中每一个探针长度为40～60bp。16. The probe composition according to item 15, wherein each probe is 40-60 bp in length.
17.根据第16项所述的探针组合物，其中每一个探针长度为45～56bp，优选50～56bp，进一步优选50bp。17. The probe composition according to item 16, wherein each probe has a length of 45-56 bp, preferably 50-56 bp, and more preferably 50 bp.
18.根据第15项所述的探针组合物，其特征在于，所述低甲基化探针包括靶向癌症特异性区域的探针Seq ID No.：118-214中的任意，靶向泛癌特异性区域的探针Seq ID No.：215-216中的任意，和靶向组织特异性区域的探针Seq ID No.：217-274中的任意。18. The probe composition according to item 15, wherein the hypomethylation probe comprises a probe targeting a cancer-specific region. Seq ID No.: any of 118-214, targeting Seq ID No. of probes for pan-cancer specific regions: any of 215-216, and Seq ID No. of probes that target tissue-specific regions: any of 217-274.
19.根据第15项所述的探针组合物，其特征在于，所述高甲基化探针包括靶向癌症特异性区域的探针Seq ID No.：275-371中的任意，靶向泛癌特异性区域的探针Seq ID No.：372-373中的任意，和靶向组织特异性区域的探针Seq ID No.：374-431中的任意。19. The probe composition according to item 15, wherein the hypermethylated probe includes a probe targeting a cancer-specific region Seq ID No.: any of 275-371, targeting pan-cancer Seq ID No. of probes for specific regions: any of 372-373, and Seq ID No. of probes for tissue-specific regions: any of 374-431.
20.一种试剂盒，其包括第1-19项中任一项所述的探针组合物。20. A kit comprising the probe composition according to any one of items 1-19.
21.一种第1-19项中任一项所述的探针组合物在制备检测食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌的试剂盒中的应用。21. A probe composition according to any one of items 1-19 is prepared for detecting esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, and cervical cancer And the application of the kit for endometrial cancer.
本申请还提供了一种利用所述探针组合物检测食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌等11种癌症的方法。The application also provides a method for detecting 11 kinds of esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer by using the probe composition Methods of cancer.
本申请同时也提供了一种利用所述试剂盒同时检测上述11种癌症的甲基化水平改变的方法。This application also provides a method for simultaneously detecting changes in the methylation level of the 11 cancers mentioned above by using the kit.
由于现有癌症检测技术的局限性，因此，需要开发一种具有以下优势的探针组合物：Due to the limitations of existing cancer detection technologies, it is necessary to develop a probe composition with the following advantages:
1液体活检属于无创肿瘤检测，对于无法获取组织样品的无症状人群和病人群体均能适用。1 Liquid biopsy is a non-invasive tumor detection, which can be applied to asymptomatic and patient groups who cannot obtain tissue samples.
2可以同时检测11种中国常见癌症的甲基化水平改变，覆盖超过80％ 的癌症发病人群。2 It can simultaneously detect changes in the methylation level of 11 common cancers in China, covering more than 80% of cancer patients.
3为增加检测的准确性，对于每一种癌症，平均测序深度超过5000X。3 In order to increase the accuracy of detection, for each cancer, the average sequencing depth exceeds 5000X.
4对于受检人，一次性可以完成所有高发癌症的筛查，提高了检测效率，每个标志物的平均价格低于市场现有单标志物的检测。4 For the subject, the screening of all high-incidence cancers can be completed at one time, which improves the detection efficiency. The average price of each marker is lower than the existing single-marker detection in the market.
5对于企业，用一个penal可以完成对主要癌症的筛选，节约了探针合成成本，可以简化实验流程，便于实验员操作。5 For companies, one penal can be used to screen for major cancers, which saves probe synthesis costs, simplifies the experimental process, and facilitates the operation of experimenters.
6原则上也可以用于癌症患者预后的癌症监测。6 In principle, it can also be used for cancer monitoring of the prognosis of cancer patients.
附图说明Description of the drawings
图1为本申请的实施操作流程Figure 1 is the implementation process of this application
具体实施方式Detailed ways
本申请提供了一种癌症基因甲基化检测的探针组合物。基于高通量测序(NGS)方法检测食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌等11种肿瘤游离DNA甲基化水平变化。其可以以非侵入的方式同时检测11种常见癌症的甲基化水平改变，灵敏性和准确定高，测序深度深且成本低，适用于无法获取组织样品的无症状人群和病人群体，以及癌症患者预后的癌症监测。This application provides a probe composition for cancer gene methylation detection. Based on high-throughput sequencing (NGS) method to detect esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and endometrial cancer and other 11 kinds of tumor free DNA methyl Changes in the level of chemistry. It can simultaneously detect changes in the methylation level of 11 common cancers in a non-invasive manner, with sensitivity and accuracy, high sequencing depth and low cost, and is suitable for asymptomatic and patient groups who cannot obtain tissue samples, as well as cancer Cancer monitoring of patient prognosis.
定义definition
除非在本文的其他地方具体限定，否则本文使用的所有其他技术和科学术语具有本申请所属领域的普通技术人员通常理解的含义。Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which this application belongs.
探针为长度在几十到几百甚至上千碱基对的单链或双链DNA，其可利用分子的变性、复性以及碱基互补配对的高度精确性，能与待测样本中互补的非标记单链DNA或RNA以氢键结合(杂交)，形成双链复合物(杂交体)。将未配对结合的探针洗去后，可用放射自显影或酶联反应等检测系统检测杂交反应结果。在本申请中，与探针互补结合或杂交的区域为特异性靶区域。多个探针组合成探针组合物。The probe is a single-stranded or double-stranded DNA with a length of tens to hundreds or even thousands of base pairs, which can take advantage of molecular denaturation, renaturation and high accuracy of base complementary pairing, and can be complementary to the sample to be tested The unlabeled single-stranded DNA or RNA is hydrogen-bonded (hybridized) to form a double-stranded complex (hybrid). After the unpaired and bound probes are washed away, detection systems such as autoradiography or enzyme-linked reaction can be used to detect the results of the hybridization reaction. In this application, the region that complementarily binds or hybridizes with the probe is the specific target region. Multiple probes are combined into a probe composition.
癌症特异性区域是指在少部分癌症种类中，与正常的对照组织相比，该区域的甲基化水平存在显著差异。A cancer-specific region refers to a significant difference in the methylation level of this region compared with normal control tissues in a small number of cancer types.
泛癌特异性区域是指在大部分癌症种类中，与正常的对照组织相比，该区域的甲基化水平存在显著差异。The pan-cancer specific region refers to a significant difference in the methylation level of this region compared with normal control tissues in most cancer types.
组织特异性区域是指该区域在特定组织中的甲基化水平与其他组织相比存在显著差异。Tissue-specific region refers to the significant difference in the methylation level of the region in a specific tissue compared with other tissues.
DNA甲基化是指发生在CpG二核苷酸中胞嘧啶上第5位碳原子的甲基化过程，作为一种对稳定的修饰状态，在DNA甲基转移酶的作用下，可随DNA的复制过程遗传给新生的子代DNA，是一种重要的表观遗传机制，DNA甲基化时，基因启动子区的甲基化可导致抑癌基因转录沉寂，因此它与肿瘤的发生关系密切。异常甲基化包括抑癌基因和DNA修复基因的高甲基化、重复序列DNA的低甲基化、某些基因的印记丢失，其与多种肿瘤的发生有关。DNA methylation refers to the methylation process that occurs at the 5th carbon atom of cytosine in CpG dinucleotides. As a stable modification state, DNA methyltransferase can follow DNA The duplication process of dna is inherited to the new generation DNA, which is an important epigenetic mechanism. When DNA is methylated, the methylation of the gene promoter region can lead to the silence of tumor suppressor gene transcription, so it is related to the occurrence of tumors. close. Abnormal methylation includes hypermethylation of tumor suppressor genes and DNA repair genes, hypomethylation of repetitive sequence DNA, and loss of imprinting of certain genes, which are related to the occurrence of a variety of tumors.
在本文中，Panel是指本文中使用的探针组合物。In this article, Panel refers to the probe composition used in this article.
以下详细描述本申请的技术方案。The technical solution of the present application is described in detail below.
在本申请的一个具体的实施方案中，一种探针组合物，包括：靶向食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌中的任意癌症的特异性区域的探针，其中，所述癌症特异性区域选自Seq ID No.：1-62中的任意；靶向泛癌特异性区域中的任意的探针，所述泛癌特异性区域选自Seq ID No.：63-64中的任意；和靶向组织特异性区域中的任意的探针，所述组织特异性区域选自Seq ID No.：65-117中的任意。所述组织特异性区域中Seq ID No.：66-67、79、81-82、97-102、116-117是食管的组织特异性靶区域，Seq ID No.：81、82、97-102是胃的组织特异性靶区域，Seq ID No.：83、87-90、110是结直肠的组织特异性靶区域。In a specific embodiment of the present application, a probe composition includes: targeted esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and A probe for a specific region of any cancer in endometrial cancer, wherein the cancer-specific region is selected from any of Seq ID No.: 1-62; a probe that targets any of the pan-cancer specific regions Needle, the pan-cancer-specific region is selected from Seq ID No.: any of 63-64; and any probes that target tissue-specific regions, the tissue-specific region is selected from Seq ID No.: Any of 65-117. Seq ID Nos.: 66-67, 79, 81-82, 97-102, 116-117 of the tissue-specific regions are tissue-specific target regions of the esophagus, Seq ID Nos.: 81, 82, 97-102 It is the tissue-specific target area of the stomach, Seq ID No.: 83, 87-90, 110 are the tissue-specific target area of the colorectal.
在本申请的一个具体的实施方案中，一种探针组合物，包括：靶向食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌中的任意癌症的特异性区域的探针，其中，所述癌症特异性区域选自Seq ID No.：1-62中的任意；靶向泛癌特异性区域中的任意的探针，所述泛癌特异性区域选自Seq ID No.：63-64中的任意；和靶向组织特异性区域中的任意的探针，所述组织特异性区域选自Seq ID No.：65-117中的任意。所述组织特异性区域中Seq ID No.：65、70-72、76、91-92、103-104 是肺的组织特异性靶区域。In a specific embodiment of the present application, a probe composition includes: targeted esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and A probe for a specific region of any cancer in endometrial cancer, wherein the cancer-specific region is selected from Seq ID No.: any of 1-62; a probe that targets any of the pan-cancer specific regions Needle, the pan-cancer-specific region is selected from Seq ID No.: any of 63-64; and any probes that target tissue-specific regions, the tissue-specific region is selected from Seq ID No.: Any of 65-117. Seq ID No. of the tissue-specific regions: 65, 70-72, 76, 91-92, 103-104 are the tissue-specific target regions of the lung.
在本申请的一个具体的实施方案中，一种探针组合物，包括：靶向食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌中的任意癌症的特异性区域的探针，其中，所述癌症特异性区域选自Seq ID No.：1-62中的任意；靶向泛癌特异性区域中的任意的探针，所述泛癌特异性区域选自Seq ID No.：63-64中的任意；和靶向组织特异性区域中的任意的探针，所述组织特异性区域选自Seq ID No.：65-117中的任意。所述组织特异性区域中Seq ID No.：73-74、107是肝的组织特异性靶区域，Seq ID No.：111-115是胰腺的组织特异性靶区域。In a specific embodiment of the present application, a probe composition includes: targeted esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and A probe for a specific region of any cancer in endometrial cancer, wherein the cancer-specific region is selected from Seq ID No.: any of 1-62; a probe that targets any of the pan-cancer specific regions Needle, the pan-cancer-specific region is selected from Seq ID No.: any of 63-64; and any probes that target tissue-specific regions, the tissue-specific region is selected from Seq ID No.: Any of 65-117. Among the tissue-specific regions, Seq ID No.: 73-74 and 107 are tissue-specific target regions of the liver, and Seq ID No.: 111-115 are tissue-specific target regions of the pancreas.
在本申请的一个具体的实施方案中，一种探针组合物，包括：靶向食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌中的任意癌症的特异性区域的探针，其中，所述癌症特异性区域选自Seq ID No.：1-62中的任意；靶向泛癌特异性区域中的任意的探针，所述泛癌特异性区域选自Seq ID No.：63-64中的任意；和靶向组织特异性区域中的任意的探针，所述组织特异性区域选自Seq ID No.：65-117中的任意。所述组织特异性区域中Seq ID No.：80、84、93、95是前列腺的组织特异性靶区域。In a specific embodiment of the present application, a probe composition includes: targeted esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and A probe for a specific region of any cancer in endometrial cancer, wherein the cancer-specific region is selected from Seq ID No.: any of 1-62; a probe that targets any of the pan-cancer specific regions Needle, the pan-cancer-specific region is selected from Seq ID No.: any of 63-64; and any probes that target tissue-specific regions, the tissue-specific region is selected from Seq ID No.: Any of 65-117. Seq ID No. in the tissue-specific region: 80, 84, 93, and 95 are tissue-specific target regions of the prostate.
在本申请的一个具体的实施方案中，一种探针组合物，包括：靶向食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌中的任意癌症的特异性区域的探针，其中，所述癌症特异性区域选自Seq ID No.：1-62中的任意；靶向泛癌特异性区域中的任意的探针，所述泛癌特异性区域选自Seq ID No.：63-64中的任意；和靶向组织特异性区域中的任意的探针，所述组织特异性区域选自Seq ID No.：65-117中的任意。所述组织特异性区域中Seq ID No.：78、84-86是乳腺的组织特异性靶区域，Seq ID No.：77、96、105是卵巢的组织特异性靶区域，Seq ID No.：68-69、75、109是宫颈的组织特异性靶区域，Seq ID No.：94、106、108是子宫内膜的组织特异性靶区域。In a specific embodiment of the present application, a probe composition includes: targeted esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and A probe for a specific region of any cancer in endometrial cancer, wherein the cancer-specific region is selected from Seq ID No.: any of 1-62; a probe that targets any of the pan-cancer specific regions Needle, the pan-cancer-specific region is selected from Seq ID No.: any of 63-64; and any probes that target tissue-specific regions, the tissue-specific region is selected from Seq ID No.: Any of 65-117. Among the tissue-specific regions, Seq ID No.: 78, 84-86 are the tissue-specific target regions of the breast, Seq ID No.: 77, 96, and 105 are the tissue-specific target regions of the ovary, and Seq ID No.: 68-69, 75, and 109 are the tissue-specific target regions of the cervix, and Seq ID No.: 94, 106, and 108 are the tissue-specific target regions of the endometrium.
在本申请的一个具体的实施方案中，上述探针组合物，包括低甲基化探针，其与经重亚硫酸盐转化的不含CG甲基化的所述特异性区域杂交，和高甲基化探针，其与经重亚硫酸盐转化的CG全部甲基化的所述特异性区域杂 交。所述低甲基化探针包括靶向癌症特异性区域的探针Seq ID No.：118-214中的任意，靶向泛癌特异性区域的探针Seq ID No.：215-216中的任意，和靶向组织特异性区域的探针Seq ID No.：217-274中的任意。所述高甲基化探针包括靶向癌症特异性区域的探针Seq ID No.：275-371中的任意，靶向泛癌特异性区域的探针Seq ID No.：372-373中的任意，和靶向组织特异性区域的探针Seq ID No.：374-431中的任意。In a specific embodiment of the present application, the above-mentioned probe composition includes a hypomethylated probe which hybridizes to the bisulfite-converted specific region without CG methylation, and high methylation A probe that hybridizes to the specific region where the bisulfite-converted CG is fully methylated. The hypomethylation probes include probes targeting cancer-specific regions Seq ID No.: any of 118-214, and probes targeting pan-cancer specific regions Seq ID No.: 215-216 Any, and the probe Seq ID No. that targets tissue-specific regions: Any of 217-274. The hypermethylation probes include probes targeting cancer-specific regions, Seq ID No.: any of 275-371, and probes targeting pan-cancer specific regions, Seq ID No.: 372-373, Seq ID No.: any of 374-431 probes targeting tissue-specific regions.
如下表1所示，其中Seq ID No.118、Seq ID No.119和Seq ID No.120均是靶向Seq ID No.1所示靶区域的低甲基化探针，Seq ID No.275、Seq ID No.276和Seq ID No.277均是靶向Seq ID No.1所示靶区域的高甲基化探针。Seq ID No.121和Seq ID No.122均是靶向Seq ID No.2所示靶区域的低甲基化探针，Seq ID No.278和Seq ID No.279均是靶向Seq ID No.2所示靶区域的高甲基化探针。Seq ID No.123和Seq ID No.124均是靶向Seq ID No.3所示靶区域的低甲基化探针，Seq ID No.280和Seq ID No.281均是靶向Seq ID No.3所示靶区域的高甲基化探针。Seq ID No.125是靶向Seq ID No.4所示靶区域的低甲基化探针，Seq ID No.282是靶向Seq ID No.4所示靶区域的高甲基化探针。Seq ID No.126和Seq ID No.127均是靶向Seq ID No.5所示靶区域的低甲基化探针，Seq ID No.283和Seq ID No.284均是靶向Seq ID No.3所示靶区域的高甲基化探针。Seq ID No.128是靶向Seq ID No.6所示靶区域的低甲基化探针，Seq ID No.285是靶向Seq ID No.6所示靶区域的高甲基化探针。Seq ID No.129是靶向Seq ID No.7所示靶区域的低甲基化探针，Seq ID No.286是靶向Seq ID No.7所示靶区域的高甲基化探针。Seq ID No.130和Seq ID No.131均是靶向Seq ID No.8所示靶区域的低甲基化探针，Seq ID No.287和Seq ID No.288均是靶向Seq ID No.8所示靶区域的高甲基化探针。Seq ID No.132、Seq ID No.133和Seq ID No.134均是靶向Seq ID No.9所示靶区域的低甲基化探针，Seq ID No.289、Seq ID No.290和Seq ID No.291均是靶向Seq ID No.9所示靶区域的高甲基化探针。Seq ID No.135是靶向Seq ID No.10所示靶区域的低甲基化探针，Seq ID No.292是靶向Seq ID No.10所示靶区域的高甲基化探针。Seq ID No.136是靶向Seq ID No.11所示靶区域的低甲基化探针，Seq ID No.293是靶向Seq ID No.11所示靶区域的高甲基化探针。Seq ID No.137是靶向Seq ID No.12所示靶区域的低甲基化探针，Seq  ID No.294是靶向Seq ID No.12所示靶区域的高甲基化探针。Seq ID No.138是靶向Seq ID No.13所示靶区域的低甲基化探针，Seq ID No.295是靶向Seq ID No.13所示靶区域的高甲基化探针。Seq ID No.139和Seq ID No.140均是靶向Seq ID No.14所示靶区域的低甲基化探针，Seq ID No.296和Seq ID No.297均是靶向Seq ID No.14所示靶区域的高甲基化探针。Seq ID No.141和Seq ID No.142均是靶向Seq ID No.15所示靶区域的低甲基化探针，Seq ID No.298和Seq ID No.299均是靶向Seq ID No.15所示靶区域的高甲基化探针。Seq ID No.143和Seq ID No.144均是靶向Seq ID No.16所示靶区域的低甲基化探针，Seq ID No.300和Seq ID No.301均是靶向Seq ID No.16所示靶区域的高甲基化探针。Seq ID No.145和Seq ID No.146均是靶向Seq ID No.17所示靶区域的低甲基化探针，Seq ID No.302和Seq ID No.303均是靶向Seq ID No.17所示靶区域的高甲基化探针。Seq ID No.147是靶向Seq ID No.18所示靶区域的低甲基化探针，Seq ID No.304是靶向Seq ID No.18所示靶区域的高甲基化探针。Seq ID No.148和Seq ID No.149均是靶向Seq ID No.19所示靶区域的低甲基化探针，Seq ID No.305和Seq ID No.306均是靶向Seq ID No.19所示靶区域的高甲基化探针。Seq ID No.150是靶向Seq ID No.20所示靶区域的低甲基化探针，Seq ID No.307是靶向Seq ID No.20所示靶区域的高甲基化探针。Seq ID No.151是靶向Seq ID No.21所示靶区域的低甲基化探针，Seq ID No.308是靶向Seq ID No.21所示靶区域的高甲基化探针。Seq ID No.152是靶向Seq ID No.22所示靶区域的低甲基化探针，Seq ID No.309是靶向Seq ID No.22所示靶区域的高甲基化探针。Seq ID No.153、Seq ID No.154和Seq ID No.155均是靶向Seq ID No.23所示靶区域的低甲基化探针，Seq ID No.310、Seq ID No.311和Seq ID No.312均是靶向Seq ID No.23所示靶区域的高甲基化探针。Seq ID No.156和Seq ID No.157均是靶向Seq ID No.24所示靶区域的低甲基化探针，Seq ID No.313和Seq ID No.314均是靶向Seq ID No.24所示靶区域的高甲基化探针。Seq ID No.158和Seq ID No.159均是靶向Seq ID No.25所示靶区域的低甲基化探针，Seq ID No.315和Seq ID No.316均是靶向Seq ID No.25所示靶区域的高甲基化探针。Seq ID No.160和Seq ID No.161均是靶向Seq ID No.26所示靶区域的低甲基化探针，Seq ID No.317和Seq ID No.318均是靶向Seq ID No.26所示靶区域的高甲基化探针。 Seq ID No.162是靶向Seq ID No.27所示靶区域的低甲基化探针，Seq ID No.319是靶向Seq ID No.27所示靶区域的高甲基化探针。Seq ID No.163和Seq ID No.164均是靶向Seq ID No.28所示靶区域的低甲基化探针，Seq ID No.320和Seq ID No.321均是靶向Seq ID No.28所示靶区域的高甲基化探针。Seq ID No.165和Seq ID No.166均是靶向Seq ID No.29所示靶区域的低甲基化探针，Seq ID No.322和Seq ID No.323均是靶向Seq ID No.29所示靶区域的高甲基化探针。Seq ID No.167是靶向Seq ID No.30所示靶区域的低甲基化探针，Seq ID No.324是靶向Seq ID No.30所示靶区域的高甲基化探针。Seq ID No.168和Seq ID No.169均是靶向Seq ID No.31所示靶区域的低甲基化探针，Seq ID No.325和Seq ID No.326均是靶向Seq ID No.31所示靶区域的高甲基化探针。Seq ID No.170是靶向Seq ID No.32所示靶区域的低甲基化探针，Seq ID No.327是靶向Seq ID No.32所示靶区域的高甲基化探针。Seq ID No.171和Seq ID No.172均是靶向Seq ID No.33所示靶区域的低甲基化探针，Seq ID No.328和Seq ID No.329均是靶向Seq ID No.33所示靶区域的高甲基化探针。Seq ID No.173是靶向Seq ID No.34所示靶区域的低甲基化探针，Seq ID No.330是靶向Seq ID No.34所示靶区域的高甲基化探针。Seq ID No.174和Seq ID No.175均是靶向Seq ID No.35所示靶区域的低甲基化探针，Seq ID No.331和Seq ID No.332均是靶向Seq ID No.35所示靶区域的高甲基化探针。Seq ID No.176是靶向Seq ID No.36所示靶区域的低甲基化探针，Seq ID No.333是靶向Seq ID No.36所示靶区域的高甲基化探针。Seq ID No.177和Seq ID No.178均是靶向Seq ID No.37所示靶区域的低甲基化探针，Seq ID No.334和Seq ID No.335均是靶向Seq ID No.37所示靶区域的高甲基化探针。Seq ID No.179和Seq ID No.180均是靶向Seq ID No.38所示靶区域的低甲基化探针，Seq ID No.336和Seq ID No.337均是靶向Seq ID No.38所示靶区域的高甲基化探针。Seq ID No.181是靶向Seq ID No.39所示靶区域的低甲基化探针，Seq ID No.338是靶向Seq ID No.39所示靶区域的高甲基化探针。Seq ID No.182和Seq ID No.183均是靶向Seq ID No.40所示靶区域的低甲基化探针，Seq ID No.339和Seq ID No.340均是靶向Seq ID No.40所示靶区域的高甲基化探针。Seq ID No.184是靶向Seq ID No.41所示靶区域的低甲基化探针，Seq ID No.341是靶向Seq ID No.41所示靶区域的 高甲基化探针。Seq ID No.185是靶向Seq ID No.42所示靶区域的低甲基化探针，Seq ID No.342是靶向Seq ID No.42所示靶区域的高甲基化探针。Seq ID No.186是靶向Seq ID No.43所示靶区域的低甲基化探针，Seq ID No.343是靶向Seq ID No.43所示靶区域的高甲基化探针。Seq ID No.187是靶向Seq ID No.44所示靶区域的低甲基化探针，Seq ID No.344是靶向Seq ID No.44所示靶区域的高甲基化探针。Seq ID No.188和Seq ID No.189均是靶向Seq ID No.45所示靶区域的低甲基化探针，Seq ID No.345和Seq ID No.346均是靶向Seq ID No.45所示靶区域的高甲基化探针。Seq ID No.190是靶向Seq ID No.46所示靶区域的低甲基化探针，Seq ID No.347是靶向Seq ID No.46所示靶区域的高甲基化探针。Seq ID No.191是靶向Seq ID No.47所示靶区域的低甲基化探针，Seq ID No.348是靶向Seq ID No.47所示靶区域的高甲基化探针。Seq ID No.192是靶向Seq ID No.48所示靶区域的低甲基化探针，Seq ID No.349是靶向Seq ID No.48所示靶区域的高甲基化探针。Seq ID No.193是靶向Seq ID No.49所示靶区域的低甲基化探针，Seq ID No.350是靶向Seq ID No.49所示靶区域的高甲基化探针。Seq ID No.194是靶向Seq ID No.50所示靶区域的低甲基化探针，Seq ID No.351是靶向Seq ID No.50所示靶区域的高甲基化探针。Seq ID No.195和Seq ID No.196均是靶向Seq ID No.51所示靶区域的低甲基化探针，Seq ID No.352和Seq ID No.353均是靶向Seq ID No.51所示靶区域的高甲基化探针。Seq ID No.197是靶向Seq ID No.52所示靶区域的低甲基化探针，Seq ID No.354是靶向Seq ID No.52所示靶区域的高甲基化探针。Seq ID No.198和Seq ID No.199均是靶向Seq ID No.53所示靶区域的低甲基化探针，Seq ID No.355和Seq ID No.356均是靶向Seq ID No.53所示靶区域的高甲基化探针。Seq ID No.200是靶向Seq ID No.54所示靶区域的低甲基化探针，Seq ID No.357是靶向Seq ID No.54所示靶区域的高甲基化探针。Seq ID No.201和Seq ID No.202均是靶向Seq ID No.55所示靶区域的低甲基化探针，Seq ID No.358和Seq ID No.359均是靶向Seq ID No.55所示靶区域的高甲基化探针。Seq ID No.203和Seq ID No.204均是靶向Seq ID No.56所示靶区域的低甲基化探针，Seq ID No.360和Seq ID No.361均是靶向Seq ID No.56所示靶区域的高甲基化探针。Seq ID No.205和Seq ID No.206均是靶向Seq ID No.57所示靶区域的低甲基化探针，Seq ID  No.362和Seq ID No.363均是靶向Seq ID No.57所示靶区域的高甲基化探针。Seq ID No.207和Seq ID No.208均是靶向Seq ID No.58所示靶区域的低甲基化探针，Seq ID No.364和Seq ID No.365均是靶向Seq ID No.58所示靶区域的高甲基化探针。Seq ID No.209和Seq ID No.210均是靶向Seq ID No.59所示靶区域的低甲基化探针，Seq ID No.366和Seq ID No.367均是靶向Seq ID No.59所示靶区域的高甲基化探针。Seq ID No.211是靶向Seq ID No.60所示靶区域的低甲基化探针，Seq ID No.368是靶向Seq ID No.60所示靶区域的高甲基化探针。Seq ID No.212和Seq ID No.213均是靶向Seq ID No.61所示靶区域的低甲基化探针，Seq ID No.369和Seq ID No.370均是靶向Seq ID No.61所示靶区域的高甲基化探针。Seq ID No.214是靶向Seq ID No.62所示靶区域的低甲基化探针，Seq ID No.371是靶向Seq ID No.62所示靶区域的高甲基化探针。Seq ID No.215是靶向Seq ID No.63所示靶区域的低甲基化探针，Seq ID No.372是靶向Seq ID No.63所示靶区域的高甲基化探针。Seq ID No.216是靶向Seq ID No.64所示靶区域的低甲基化探针，Seq ID No.373是靶向Seq ID No.64所示靶区域的高甲基化探针。Seq ID No.217是靶向Seq ID No.65所示靶区域的低甲基化探针，Seq ID No.374是靶向Seq ID No.65所示靶区域的高甲基化探针。Seq ID No.218是靶向Seq ID No.66所示靶区域的低甲基化探针，Seq ID No.375是靶向Seq ID No.66所示靶区域的高甲基化探针。Seq ID No.219是靶向Seq ID No.67所示靶区域的低甲基化探针，Seq ID No.376是靶向Seq ID No.67所示靶区域的高甲基化探针。Seq ID No.220是靶向Seq ID No.68所示靶区域的低甲基化探针，Seq ID No.377是靶向Seq ID No.68所示靶区域的高甲基化探针。Seq ID No.221是靶向Seq ID No.69所示靶区域的低甲基化探针，Seq ID No.378是靶向Seq ID No.69所示靶区域的高甲基化探针。Seq ID No.222是靶向Seq ID No.70所示靶区域的低甲基化探针，Seq ID No.379是靶向Seq ID No.70所示靶区域的高甲基化探针。Seq ID No.223是靶向Seq ID No.71所示靶区域的低甲基化探针，Seq ID No.380是靶向Seq ID No.71所示靶区域的高甲基化探针。Seq ID No.224是靶向Seq ID No.72所示靶区域的低甲基化探针，Seq ID No.381是靶向Seq ID No.72所示靶区域的高甲基化探针。Seq ID No.225是靶向Seq ID No.73所示靶区域的低甲基化探针，Seq ID No.382是靶向Seq ID No.73所示靶区 域的高甲基化探针。Seq ID No.226是靶向Seq ID No.74所示靶区域的低甲基化探针，Seq ID No.383是靶向Seq ID No.74所示靶区域的高甲基化探针。Seq ID No.227是靶向Seq ID No.75所示靶区域的低甲基化探针，Seq ID No.384是靶向Seq ID No.75所示靶区域的高甲基化探针。Seq ID No.228和Seq ID No.229均是靶向Seq ID No.76所示靶区域的低甲基化探针，Seq ID No.385和Seq ID No.386均是靶向Seq ID No.76所示靶区域的高甲基化探针。Seq ID No.230是靶向Seq ID No.77所示靶区域的低甲基化探针，Seq ID No.387是靶向Seq ID No.77所示靶区域的高甲基化探针。Seq ID No.231是靶向Seq ID No.78所示靶区域的低甲基化探针，Seq ID No.388是靶向Seq ID No.78所示靶区域的高甲基化探针。Seq ID No.232是靶向Seq ID No.79所示靶区域的低甲基化探针，Seq ID No.389是靶向Seq ID No.79所示靶区域的高甲基化探针。Seq ID No.233是靶向Seq ID No.80所示靶区域的低甲基化探针，Seq ID No.390是靶向Seq ID No.80所示靶区域的高甲基化探针。Seq ID No.234是靶向Seq ID No.81所示靶区域的低甲基化探针，Seq ID No.391是靶向Seq ID No.81所示靶区域的高甲基化探针。Seq ID No.235是靶向Seq ID No.82所示靶区域的低甲基化探针，Seq ID No.392是靶向Seq ID No.82所示靶区域的高甲基化探针。Seq ID No.236是靶向Seq ID No.83所示靶区域的低甲基化探针，Seq ID No.393是靶向Seq ID No.83所示靶区域的高甲基化探针。Seq ID No.237是靶向Seq ID No.84所示靶区域的低甲基化探针，Seq ID No.394是靶向Seq ID No.84所示靶区域的高甲基化探针。Seq ID No.238是靶向Seq ID No.85所示靶区域的低甲基化探针，Seq ID No.395是靶向Seq ID No.85所示靶区域的高甲基化探针。Seq ID No.239是靶向Seq ID No.86所示靶区域的低甲基化探针，Seq ID No.396是靶向Seq ID No.86所示靶区域的高甲基化探针。Seq ID No.240是靶向Seq ID No.87所示靶区域的低甲基化探针，Seq ID No.397是靶向Seq ID No.87所示靶区域的高甲基化探针。Seq ID No.241是靶向Seq ID No.88所示靶区域的低甲基化探针，Seq ID No.398是靶向Seq ID No.88所示靶区域的高甲基化探针。Seq ID No.242是靶向Seq ID No.89所示靶区域的低甲基化探针，Seq ID No.399是靶向Seq ID No.89所示靶区域的高甲基化探针。Seq ID No.243是靶向Seq ID No.90所示靶区域的低甲基化探针，Seq ID No.400是靶向Seq ID No.90所示靶区 域的高甲基化探针。Seq ID No.244是靶向Seq ID No.91所示靶区域的低甲基化探针，Seq ID No.401是靶向Seq ID No.91所示靶区域的高甲基化探针。Seq ID No.245是靶向Seq ID No.92所示靶区域的低甲基化探针，Seq ID No.402是靶向Seq ID No.92所示靶区域的高甲基化探针。Seq ID No.246是靶向Seq ID No.93所示靶区域的低甲基化探针，Seq ID No.403是靶向Seq ID No.93所示靶区域的高甲基化探针。Seq ID No.247是靶向Seq ID No.94所示靶区域的低甲基化探针，Seq ID No.404是靶向Seq ID No.94所示靶区域的高甲基化探针。Seq ID No.248是靶向Seq ID No.95所示靶区域的低甲基化探针，Seq ID No.405是靶向Seq ID No.95所示靶区域的高甲基化探针。Seq ID No.249是靶向Seq ID No.96所示靶区域的低甲基化探针，Seq ID No.406是靶向Seq ID No.96所示靶区域的高甲基化探针。Seq ID No.250是靶向Seq ID No.97所示靶区域的低甲基化探针，Seq ID No.407是靶向Seq ID No.97所示靶区域的高甲基化探针。Seq ID No.251是靶向Seq ID No.98所示靶区域的低甲基化探针，Seq ID No.408是靶向Seq ID No.98所示靶区域的高甲基化探针。Seq ID No.252是靶向Seq ID No.99所示靶区域的低甲基化探针，Seq ID No.409是靶向Seq ID No.99所示靶区域的高甲基化探针。Seq ID No.253是靶向Seq ID No.100所示靶区域的低甲基化探针，Seq ID No.410是靶向Seq ID No.100所示靶区域的高甲基化探针。Seq ID No.254是靶向Seq ID No.101所示靶区域的低甲基化探针，Seq ID No.411是靶向Seq ID No.101所示靶区域的高甲基化探针。Seq ID No.255和Seq ID No.256均是靶向Seq ID No.102所示靶区域的低甲基化探针，Seq ID No.412和Seq ID No.413均是靶向Seq ID No.102所示靶区域的高甲基化探针。Seq ID No.257是靶向Seq ID No.103所示靶区域的低甲基化探针，Seq ID No.414是靶向Seq ID No.103所示靶区域的高甲基化探针。Seq ID No.258是靶向Seq ID No.104所示靶区域的低甲基化探针，Seq ID No.415是靶向Seq ID No.104所示靶区域的高甲基化探针。Seq ID No.259是靶向Seq ID No.105所示靶区域的低甲基化探针，Seq ID No.416是靶向Seq ID No.105所示靶区域的高甲基化探针。Seq ID No.260是靶向Seq ID No.106所示靶区域的低甲基化探针，Seq ID No.417是靶向Seq ID No.106所示靶区域的高甲基化探针。Seq ID No.261是靶向Seq ID No.107所示靶区域的低甲基化探针，Seq ID No.418是靶向Seq ID No.107 所示靶区域的高甲基化探针。Seq ID No.262是靶向Seq ID No.108所示靶区域的低甲基化探针，Seq ID No.419是靶向Seq ID No.108所示靶区域的高甲基化探针。Seq ID No.263是靶向Seq ID No.109所示靶区域的低甲基化探针，Seq ID No.420是靶向Seq ID No.109所示靶区域的高甲基化探针。Seq ID No.264、Seq ID No.265和Seq ID No.266均是靶向Seq ID No.110所示靶区域的低甲基化探针，Seq ID No.421、Seq ID No.422和Seq ID No.423均是靶向Seq ID No.110所示靶区域的高甲基化探针。Seq ID No.267是靶向Seq ID No.111所示靶区域的低甲基化探针，Seq ID No.424是靶向Seq ID No.111所示靶区域的高甲基化探针。Seq ID No.268是靶向Seq ID No.112所示靶区域的低甲基化探针，Seq ID No.425是靶向Seq ID No.112所示靶区域的高甲基化探针。Seq ID No.269是靶向Seq ID No.113所示靶区域的低甲基化探针，Seq ID No.426是靶向Seq ID No.113所示靶区域的高甲基化探针。Seq ID No.270和Seq ID No.271均是靶向Seq ID No.114所示靶区域的低甲基化探针，Seq ID No.427和Seq ID No.428均是靶向Seq ID No.114所示靶区域的高甲基化探针。Seq ID No.272是靶向Seq ID No.115所示靶区域的低甲基化探针，Seq ID No.429是靶向Seq ID No.115所示靶区域的高甲基化探针。Seq ID No.273是靶向Seq ID No.116所示靶区域的低甲基化探针，Seq ID No.430是靶向Seq ID No.116所示靶区域的高甲基化探针。Seq ID No.274是靶向Seq ID No.117所示靶区域的低甲基化探针，Seq ID No.431是靶向Seq ID No.117所示靶区域的高甲基化探针。表1中给出了探针所靶向的靶序列。As shown in Table 1 below, Seq ID No. 118, Seq ID No. 119 and Seq ID No. 120 are all hypomethylated probes that target the target region shown in Seq ID No. 1, Seq ID No. 275 , Seq ID No. 276 and Seq ID No. 277 are all hypermethylated probes that target the target region shown in Seq ID No. 1. Seq ID No. 121 and Seq ID No. 122 are hypomethylation probes that target the target region shown in Seq ID No. 2, and Seq ID No. 278 and Seq ID No. 279 are both targeted to Seq ID No. .2 Hypermethylated probes in the target region shown. Seq ID No. 123 and Seq ID No. 124 are hypomethylation probes that target the target region shown in Seq ID No. 3, and Seq ID No. 280 and Seq ID No. 281 are both targeted to Seq ID No. .3 Hypermethylated probes in the target region shown. Seq ID No. 125 is a hypomethylated probe that targets the target region shown in Seq ID No. 4, and Seq ID No. 282 is a hypermethylated probe that targets the target region shown in Seq ID No. 4. Seq ID No. 126 and Seq ID No. 127 are both hypomethylated probes that target the target region shown in Seq ID No. 5, and Seq ID No. 283 and Seq ID No. 284 are both targeted to Seq ID No. .3 Hypermethylated probes in the target region shown. Seq ID No. 128 is a hypomethylated probe targeting the target region shown in Seq ID No. 6, and Seq ID No. 285 is a hypermethylated probe targeting the target region shown in Seq ID No. 6. Seq ID No. 129 is a hypomethylated probe targeting the target region shown in Seq ID No. 7, and Seq ID No. 286 is a hypermethylated probe targeting the target region shown in Seq ID No. 7. Seq ID No. 130 and Seq ID No. 131 are both hypomethylated probes that target the target region shown in Seq ID No. 8, and Seq ID No. 287 and Seq ID No. 288 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .8. Seq ID No. 132, Seq ID No. 133 and Seq ID No. 134 are all hypomethylated probes that target the target region shown in Seq ID No. 9, Seq ID No. 289, Seq ID No. 290 and Seq ID No. 291 is a hypermethylated probe that targets the target region shown in Seq ID No. 9. Seq ID No.135 is a hypomethylated probe targeting the target region shown in Seq ID No.10, and Seq ID No.292 is a hypermethylated probe targeting the target region shown in Seq ID No.10. Seq ID No.136 is a hypomethylated probe targeting the target region shown in Seq ID No.11, and Seq ID No.293 is a hypermethylated probe targeting the target region shown in Seq ID No.11. Seq ID No.137 is a hypomethylated probe targeting the target region shown in Seq ID No.12, and Seq ID No.294 is a hypermethylated probe targeting the target region shown in Seq ID No.12. Seq ID No.138 is a hypomethylated probe targeting the target region shown in Seq ID No.13, and Seq ID No.295 is a hypermethylated probe targeting the target region shown in Seq ID No.13. Seq ID No. 139 and Seq ID No. 140 are both hypomethylated probes that target the target region shown in Seq ID No. 14, and Seq ID No. 296 and Seq ID No. 297 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .14. Seq ID No. 141 and Seq ID No. 142 are hypomethylated probes that target the target region shown in Seq ID No. 15, and Seq ID No. 298 and Seq ID No. 299 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .15. Seq ID No. 143 and Seq ID No. 144 are hypomethylated probes that target the target region shown in Seq ID No. 16, and Seq ID No. 300 and Seq ID No. 301 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .16. Seq ID No. 145 and Seq ID No. 146 are both hypomethylated probes that target the target region shown in Seq ID No. 17, and Seq ID No. 302 and Seq ID No. 303 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .17. Seq ID No. 147 is a hypomethylated probe targeting the target region shown in Seq ID No. 18, and Seq ID No. 304 is a hypermethylated probe targeting the target region shown in Seq ID No. 18. Seq ID No. 148 and Seq ID No. 149 are both hypomethylated probes that target the target region shown in Seq ID No. 19, and Seq ID No. 305 and Seq ID No. 306 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .19. Seq ID No. 150 is a hypomethylated probe targeting the target region shown in Seq ID No. 20, and Seq ID No. 307 is a hypermethylated probe targeting the target region shown in Seq ID No. 20. Seq ID No. 151 is a hypomethylated probe targeting the target region shown in Seq ID No. 21, and Seq ID No. 308 is a hypermethylated probe targeting the target region shown in Seq ID No. 21. Seq ID No. 152 is a hypomethylated probe targeting the target region shown in Seq ID No. 22, and Seq ID No. 309 is a hypermethylated probe targeting the target region shown in Seq ID No. 22. Seq ID No. 153, Seq ID No. 154, and Seq ID No. 155 are all hypomethylated probes that target the target region shown in Seq ID No. 23, Seq ID No. 310, Seq ID No. 311, and Seq ID No. 311. Seq ID No. 312 is a hypermethylated probe that targets the target region shown in Seq ID No. 23. Seq ID No. 156 and Seq ID No. 157 are hypomethylated probes that target the target region shown in Seq ID No. 24, and Seq ID No. 313 and Seq ID No. 314 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .24. Seq ID No. 158 and Seq ID No. 159 are both hypomethylated probes that target the target region shown in Seq ID No. 25, and Seq ID No. 315 and Seq ID No. 316 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .25. Seq ID No. 160 and Seq ID No. 161 are hypomethylation probes that target the target region shown in Seq ID No. 26, and Seq ID No. 317 and Seq ID No. 318 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .26. Seq ID No.162 is a hypomethylated probe targeting the target region shown in Seq ID No.27, and Seq ID No.319 is a hypermethylated probe targeting the target region shown in Seq ID No.27. Seq ID No. 163 and Seq ID No. 164 are hypomethylation probes that target the target region shown in Seq ID No. 28, and Seq ID No. 320 and Seq ID No. 321 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .28. Seq ID No. 165 and Seq ID No. 166 are both hypomethylated probes that target the target region shown in Seq ID No. 29, and Seq ID No. 322 and Seq ID No. 323 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .29. Seq ID No. 167 is a hypomethylated probe that targets the target region shown in Seq ID No. 30, and Seq ID No. 324 is a hypermethylated probe that targets the target region shown in Seq ID No. 30. Seq ID No. 168 and Seq ID No. 169 are both hypomethylated probes that target the target region shown in Seq ID No. 31, and Seq ID No. 325 and Seq ID No. 326 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .31. Seq ID No. 170 is a hypomethylated probe targeting the target region shown in Seq ID No. 32, and Seq ID No. 327 is a hypermethylated probe targeting the target region shown in Seq ID No. 32. Seq ID No. 171 and Seq ID No. 172 are both hypomethylated probes that target the target region shown in Seq ID No. 33, and Seq ID No. 328 and Seq ID No. 329 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .33. Seq ID No. 173 is a hypomethylated probe targeting the target region shown in Seq ID No. 34, and Seq ID No. 330 is a hypermethylated probe targeting the target region shown in Seq ID No. 34. Seq ID No. 174 and Seq ID No. 175 are hypomethylated probes that target the target region shown in Seq ID No. 35, and Seq ID No. 331 and Seq ID No. 332 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .35. Seq ID No. 176 is a hypomethylated probe targeting the target region shown in Seq ID No. 36, and Seq ID No. 333 is a hypermethylated probe targeting the target region shown in Seq ID No. 36. Seq ID No. 177 and Seq ID No. 178 are hypomethylated probes that target the target region shown in Seq ID No. 37, and Seq ID No. 334 and Seq ID No. 335 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .37. Seq ID No. 179 and Seq ID No. 180 are hypomethylated probes that target the target region shown in Seq ID No. 38, and Seq ID No. 336 and Seq ID No. 337 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .38. Seq ID No. 181 is a hypomethylated probe that targets the target region shown in Seq ID No. 39, and Seq ID No. 338 is a hypermethylated probe that targets the target region shown in Seq ID No. 39. Seq ID No. 182 and Seq ID No. 183 are hypomethylated probes that target the target region shown in Seq ID No. 40, and Seq ID No. 339 and Seq ID No. 340 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .40. Seq ID No. 184 is a hypomethylated probe targeting the target region shown in Seq ID No. 41, and Seq ID No. 341 is a hypermethylated probe targeting the target region shown in Seq ID No. 41. Seq ID No.185 is a hypomethylated probe targeting the target region shown in Seq ID No.42, and Seq ID No.342 is a hypermethylated probe targeting the target region shown in Seq ID No.42. Seq ID No. 186 is a hypomethylated probe targeting the target region shown in Seq ID No. 43, and Seq ID No. 343 is a hypermethylated probe targeting the target region shown in Seq ID No. 43. Seq ID No. 187 is a hypomethylated probe targeting the target region shown in Seq ID No. 44, and Seq ID No. 344 is a hypermethylated probe targeting the target region shown in Seq ID No. 44. Seq ID No. 188 and Seq ID No. 189 are both hypomethylated probes that target the target region shown in Seq ID No. 45, and Seq ID No. 345 and Seq ID No. 346 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .45. Seq ID No. 190 is a hypomethylated probe that targets the target region shown in Seq ID No. 46, and Seq ID No. 347 is a hypermethylated probe that targets the target region shown in Seq ID No. 46. Seq ID No. 191 is a hypomethylated probe targeting the target region shown in Seq ID No. 47, and Seq ID No. 348 is a hypermethylated probe targeting the target region shown in Seq ID No. 47. Seq ID No. 192 is a hypomethylated probe targeting the target region shown in Seq ID No. 48, and Seq ID No. 349 is a hypermethylated probe targeting the target region shown in Seq ID No. 48. Seq ID No. 193 is a hypomethylated probe targeting the target region shown in Seq ID No. 49, and Seq ID No. 350 is a hypermethylated probe targeting the target region shown in Seq ID No. 49. Seq ID No. 194 is a hypomethylated probe targeting the target region shown in Seq ID No. 50, and Seq ID No. 351 is a hypermethylated probe targeting the target region shown in Seq ID No. 50. Seq ID No. 195 and Seq ID No. 196 are hypomethylation probes that target the target region shown in Seq ID No. 51, and Seq ID No. 352 and Seq ID No. 353 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .51. Seq ID No.197 is a hypomethylated probe targeting the target region shown in Seq ID No.52, and Seq ID No.354 is a hypermethylated probe targeting the target region shown in Seq ID No.52. Seq ID No. 198 and Seq ID No. 199 are hypomethylated probes that target the target region shown in Seq ID No. 53, and Seq ID No. 355 and Seq ID No. 356 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .53. Seq ID No. 200 is a hypomethylated probe targeting the target region shown in Seq ID No. 54, and Seq ID No. 357 is a hypermethylated probe targeting the target region shown in Seq ID No. 54. Seq ID No. 201 and Seq ID No. 202 are hypomethylated probes that target the target region shown in Seq ID No. 55, and Seq ID No. 358 and Seq ID No. 359 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .55. Seq ID No. 203 and Seq ID No. 204 are hypomethylated probes that target the target region shown in Seq ID No. 56, and Seq ID No. 360 and Seq ID No. 361 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .56. Seq ID No. 205 and Seq ID No. 206 are both hypomethylated probes that target the target region shown in Seq ID No. 57, and Seq ID No. 362 and Seq ID No. 363 are both targeted to Seq ID No. A hypermethylated probe in the target region shown in .57. Seq ID No. 207 and Seq ID No. 208 are hypomethylated probes that target the target region shown in Seq ID No. 58, and Seq ID No. 364 and Seq ID No. 365 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .58. Seq ID No. 209 and Seq ID No. 210 are hypomethylated probes that target the target region shown in Seq ID No. 59, and Seq ID No. 366 and Seq ID No. 367 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .59. Seq ID No. 211 is a hypomethylated probe targeting the target region shown in Seq ID No. 60, and Seq ID No. 368 is a hypermethylated probe targeting the target region shown in Seq ID No. 60. Seq ID No. 212 and Seq ID No. 213 are hypomethylated probes that target the target region shown in Seq ID No. 61, and Seq ID No. 369 and Seq ID No. 370 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .61. Seq ID No. 214 is a hypomethylated probe targeting the target region shown in Seq ID No. 62, and Seq ID No. 371 is a hypermethylated probe targeting the target region shown in Seq ID No. 62. Seq ID No. 215 is a hypomethylated probe targeting the target region shown in Seq ID No. 63, and Seq ID No. 372 is a hypermethylated probe targeting the target region shown in Seq ID No. 63. Seq ID No.216 is a hypomethylated probe targeting the target region shown in Seq ID No.64, and Seq ID No.373 is a hypermethylated probe targeting the target region shown in Seq ID No.64. Seq ID No. 217 is a hypomethylated probe targeting the target region shown in Seq ID No. 65, and Seq ID No. 374 is a hypermethylated probe targeting the target region shown in Seq ID No. 65. Seq ID No. 218 is a hypomethylated probe targeting the target region shown in Seq ID No. 66, and Seq ID No. 375 is a hypermethylated probe targeting the target region shown in Seq ID No. 66. Seq ID No. 219 is a hypomethylated probe targeting the target region shown in Seq ID No. 67, and Seq ID No. 376 is a hypermethylated probe targeting the target region shown in Seq ID No. 67. Seq ID No. 220 is a hypomethylated probe targeting the target region shown in Seq ID No. 68, and Seq ID No. 377 is a hypermethylated probe targeting the target region shown in Seq ID No. 68. Seq ID No. 221 is a hypomethylated probe that targets the target region shown in Seq ID No. 69, and Seq ID No. 378 is a hypermethylated probe that targets the target region shown in Seq ID No. 69. Seq ID No. 222 is a hypomethylated probe targeting the target region shown in Seq ID No. 70, and Seq ID No. 379 is a hypermethylated probe targeting the target region shown in Seq ID No. 70. Seq ID No. 223 is a hypomethylated probe targeting the target region shown in Seq ID No. 71, and Seq ID No. 380 is a hypermethylated probe targeting the target region shown in Seq ID No. 71. Seq ID No. 224 is a hypomethylated probe targeting the target region shown in Seq ID No. 72, and Seq ID No. 381 is a hypermethylated probe targeting the target region shown in Seq ID No. 72. Seq ID No. 225 is a hypomethylated probe targeting the target region shown in Seq ID No. 73, and Seq ID No. 382 is a hypermethylated probe targeting the target region shown in Seq ID No. 73. Seq ID No. 226 is a hypomethylated probe targeting the target region shown in Seq ID No. 74, and Seq ID No. 383 is a hypermethylated probe targeting the target region shown in Seq ID No. 74. Seq ID No. 227 is a hypomethylated probe targeting the target region shown in Seq ID No. 75, and Seq ID No. 384 is a hypermethylated probe targeting the target region shown in Seq ID No. 75. Seq ID No. 228 and Seq ID No. 229 are both hypomethylated probes that target the target region shown in Seq ID No. 76, and Seq ID No. 385 and Seq ID No. 386 are both targeted to Seq ID No. A hypermethylated probe in the target region shown in .76. Seq ID No. 230 is a hypomethylated probe that targets the target region shown in Seq ID No. 77, and Seq ID No. 387 is a hypermethylated probe that targets the target region shown in Seq ID No. 77. Seq ID No. 231 is a hypomethylated probe targeting the target region shown in Seq ID No. 78, and Seq ID No. 388 is a hypermethylated probe targeting the target region shown in Seq ID No. 78. Seq ID No. 232 is a hypomethylated probe that targets the target region shown in Seq ID No. 79, and Seq ID No. 389 is a hypermethylated probe that targets the target region shown in Seq ID No. 79. Seq ID No. 233 is a hypomethylated probe targeting the target region shown in Seq ID No. 80, and Seq ID No. 390 is a hypermethylated probe targeting the target region shown in Seq ID No. 80. Seq ID No. 234 is a hypomethylated probe targeting the target region shown in Seq ID No. 81, and Seq ID No. 391 is a hypermethylated probe targeting the target region shown in Seq ID No. 81. Seq ID No. 235 is a hypomethylated probe that targets the target region shown in Seq ID No. 82, and Seq ID No. 392 is a hypermethylated probe that targets the target region shown in Seq ID No. 82. Seq ID No. 236 is a hypomethylated probe targeting the target region shown in Seq ID No. 83, and Seq ID No. 393 is a hypermethylated probe targeting the target region shown in Seq ID No. 83. Seq ID No. 237 is a hypomethylated probe targeting the target region shown in Seq ID No. 84, and Seq ID No. 394 is a hypermethylated probe targeting the target region shown in Seq ID No. 84. Seq ID No. 238 is a hypomethylated probe targeting the target region shown in Seq ID No. 85, and Seq ID No. 395 is a hypermethylated probe targeting the target region shown in Seq ID No. 85. Seq ID No. 239 is a hypomethylated probe targeting the target region shown in Seq ID No. 86, and Seq ID No. 396 is a hypermethylated probe targeting the target region shown in Seq ID No. 86. Seq ID No. 240 is a hypomethylated probe that targets the target region shown in Seq ID No. 87, and Seq ID No. 397 is a hypermethylated probe that targets the target region shown in Seq ID No. 87. Seq ID No. 241 is a hypomethylated probe targeting the target region shown in Seq ID No. 88, and Seq ID No. 398 is a hypermethylated probe targeting the target region shown in Seq ID No. 88. Seq ID No. 242 is a hypomethylated probe targeting the target region shown in Seq ID No. 89, and Seq ID No. 399 is a hypermethylated probe targeting the target region shown in Seq ID No. 89. Seq ID No. 243 is a hypomethylated probe that targets the target region shown in Seq ID No. 90, and Seq ID No. 400 is a hypermethylated probe that targets the target region shown in Seq ID No. 90. Seq ID No. 244 is a hypomethylated probe targeting the target region shown in Seq ID No. 91, and Seq ID No. 401 is a hypermethylated probe targeting the target region shown in Seq ID No. 91. Seq ID No. 245 is a hypomethylated probe targeting the target region shown in Seq ID No. 92, and Seq ID No. 402 is a hypermethylated probe targeting the target region shown in Seq ID No. 92. Seq ID No. 246 is a hypomethylated probe targeting the target region shown in Seq ID No. 93, and Seq ID No. 403 is a hypermethylated probe targeting the target region shown in Seq ID No. 93. Seq ID No. 247 is a hypomethylated probe targeting the target region shown in Seq ID No. 94, and Seq ID No. 404 is a hypermethylated probe targeting the target region shown in Seq ID No. 94. Seq ID No. 248 is a hypomethylated probe targeting the target region shown in Seq ID No. 95, and Seq ID No. 405 is a hypermethylated probe targeting the target region shown in Seq ID No. 95. Seq ID No. 249 is a hypomethylated probe that targets the target region shown in Seq ID No. 96, and Seq ID No. 406 is a hypermethylated probe that targets the target region shown in Seq ID No. 96. Seq ID No. 250 is a hypomethylated probe targeting the target region shown in Seq ID No. 97, and Seq ID No. 407 is a hypermethylated probe targeting the target region shown in Seq ID No. 97. Seq ID No. 251 is a hypomethylated probe targeting the target region shown in Seq ID No. 98, and Seq ID No. 408 is a hypermethylated probe targeting the target region shown in Seq ID No. 98. Seq ID No. 252 is a hypomethylated probe that targets the target region shown in Seq ID No. 99, and Seq ID No. 409 is a hypermethylated probe that targets the target region shown in Seq ID No. 99. Seq ID No.253 is a hypomethylated probe targeting the target region shown in Seq ID No.100, and Seq ID No.410 is a hypermethylated probe targeting the target region shown in Seq ID No.100. Seq ID No. 254 is a hypomethylated probe targeting the target region shown in Seq ID No. 101, and Seq ID No. 411 is a hypermethylated probe targeting the target region shown in Seq ID No. 101. Seq ID No. 255 and Seq ID No. 256 are both hypomethylated probes that target the target region shown in Seq ID No. 102, and Seq ID No. 412 and Seq ID No. 413 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .102. Seq ID No. 257 is a hypomethylated probe targeting the target region shown in Seq ID No. 103, and Seq ID No. 414 is a hypermethylated probe targeting the target region shown in Seq ID No. 103. Seq ID No. 258 is a hypomethylated probe targeting the target region shown in Seq ID No. 104, and Seq ID No. 415 is a hypermethylated probe targeting the target region shown in Seq ID No. 104. Seq ID No. 259 is a hypomethylated probe targeting the target region shown in Seq ID No. 105, and Seq ID No. 416 is a hypermethylated probe targeting the target region shown in Seq ID No. 105. Seq ID No. 260 is a hypomethylated probe targeting the target region shown in Seq ID No. 106, and Seq ID No. 417 is a hypermethylated probe targeting the target region shown in Seq ID No. 106. Seq ID No. 261 is a hypomethylated probe targeting the target region shown in Seq ID No. 107, and Seq ID No. 418 is a hypermethylated probe targeting the target region shown in Seq ID No. 107. Seq ID No. 262 is a hypomethylated probe targeting the target region shown in Seq ID No. 108, and Seq ID No. 419 is a hypermethylated probe targeting the target region shown in Seq ID No. 108. Seq ID No. 263 is a hypomethylated probe targeting the target region shown in Seq ID No. 109, and Seq ID No. 420 is a hypermethylated probe targeting the target region shown in Seq ID No. 109. Seq ID No.264, Seq ID No.265 and Seq ID No.266 are all hypomethylated probes that target the target region shown in Seq ID No.110, Seq ID No.421, Seq ID No.422 and Seq ID No. 423 are all hypermethylated probes that target the target region shown in Seq ID No. 110. Seq ID No. 267 is a hypomethylated probe targeting the target region shown in Seq ID No. 111, and Seq ID No. 424 is a hypermethylated probe targeting the target region shown in Seq ID No. 111. Seq ID No. 268 is a hypomethylated probe targeting the target region shown in Seq ID No. 112, and Seq ID No. 425 is a hypermethylated probe targeting the target region shown in Seq ID No. 112. Seq ID No. 269 is a hypomethylated probe targeting the target region shown in Seq ID No. 113, and Seq ID No. 426 is a hypermethylated probe targeting the target region shown in Seq ID No. 113. Seq ID No. 270 and Seq ID No. 271 are both hypomethylated probes that target the target region shown in Seq ID No. 114, and Seq ID No. 427 and Seq ID No. 428 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .114. Seq ID No. 272 is a hypomethylated probe targeting the target region shown in Seq ID No. 115, and Seq ID No. 429 is a hypermethylated probe targeting the target region shown in Seq ID No. 115. Seq ID No. 273 is a hypomethylated probe targeting the target region shown in Seq ID No. 116, and Seq ID No. 430 is a hypermethylated probe targeting the target region shown in Seq ID No. 116. Seq ID No.274 is a hypomethylated probe targeting the target region shown in Seq ID No.117, and Seq ID No.431 is a hypermethylated probe targeting the target region shown in Seq ID No.117. Table 1 shows the target sequence targeted by the probe.
在本申请的一个具体的实施方案中，上述探针组合物中每一个探针的长度为40～60bp，优选为45～56bp，优选为50～56bp，进一步优选为50bp。In a specific embodiment of the present application, the length of each probe in the above-mentioned probe composition is 40-60 bp, preferably 45-56 bp, preferably 50-56 bp, and more preferably 50 bp.
本申请还提供了一种试剂盒。The application also provides a kit.
在本申请的一个具体的实施方案中，所述试剂盒包括上述任一实施方案所述的探针组合物。In a specific embodiment of the present application, the kit includes the probe composition described in any of the above embodiments.
本申请还提供了探针组合物的用途。The application also provides the use of the probe composition.
在本申请的一个具体的实施方案中，上述探针组合物用于制备检测食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌的试剂盒。In a specific embodiment of the present application, the above-mentioned probe composition is used to prepare and detect esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and intrauterine cancer. Membrane cancer kit.
本申请还提供了一种芯片。The application also provides a chip.
在本申请的一个具体的实施方案中，所述芯片上固定有上述任一实施方案所述的探针组合物。In a specific embodiment of the present application, the probe composition described in any of the above embodiments is immobilized on the chip.
本申请还提供了一种利用所述探针组合物检测食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌等11种癌症的方法。The application also provides a method for detecting 11 kinds of esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer by using the probe composition Methods of cancer.
在本申请的一个具体的实施方案中，所述检测方法采用杂交捕获的方式富集cfDNA，并用NGS技术检测与癌症高度相关的甲基化位点。覆盖了我国发病率最高的六个恶性肿瘤(肺癌、胃癌、结直肠癌、肝癌、乳腺癌和食管癌)，以及恶性程度极高的胰腺癌。同时包括其他三种女性肿瘤和一种男性肿瘤(宫颈癌、卵巢癌、子宫内膜癌和前列腺癌)。最终，根据检测血浆cfDNA中基因甲基化变化水平，为多癌早筛和早诊提供信息。In a specific embodiment of the present application, the detection method uses hybrid capture to enrich cfDNA, and uses NGS technology to detect methylation sites that are highly related to cancer. It covers the six malignant tumors with the highest incidence in my country (lung cancer, gastric cancer, colorectal cancer, liver cancer, breast cancer and esophageal cancer), as well as pancreatic cancer, which is extremely malignant. It also includes three other female tumors and one male tumor (cervical cancer, ovarian cancer, endometrial cancer, and prostate cancer). Finally, based on the detection of gene methylation changes in plasma cfDNA, it provides information for early screening and early diagnosis of multiple cancers.
本申请同时也提供了一种利用所述试剂盒同时检测上述11种癌症的甲基化水平改变的方法。This application also provides a method for simultaneously detecting changes in the methylation level of the 11 cancers mentioned above by using the kit.
具体来说，本申请涉及受试者11种癌症体外检测方法，包括以下步骤：采集受试者样品；提取纯化所述样品中的DNA；针对纯化的DNA样品构建用于测序的DNA文库；用重亚硫酸盐转化所述构建的DNA文库；预PCR扩增所述经重亚硫酸盐转化的DNA文库；利用探针组合物对经预PCR扩增的样品进行杂交捕获；利用PCR扩增经杂交捕获后的产物；对PCR扩增后的经杂交捕获后的产物进行高通量二代测序；对测序数据进行分析，确定样本的甲基化水平；基于所述样本的甲基化水平判读所述患者的患病情况。Specifically, this application relates to 11 in vitro cancer detection methods for subjects, including the following steps: collecting a sample of the subject; extracting and purifying the DNA in the sample; constructing a DNA library for sequencing based on the purified DNA sample; Bisulfite transforms the constructed DNA library; pre-PCR amplifies the bisulfite-converted DNA library; uses the probe composition to hybridize and capture the sample amplified by pre-PCR; Products captured by hybridization; perform high-throughput next-generation sequencing on the products amplified by PCR and captured by hybridization; analyze the sequencing data to determine the methylation level of the sample; judge based on the methylation level of the sample The condition of the patient.
具体来说，所述受试者疑似患有癌症。具体来说，采集受试者的样品为血浆样品。具体来说，所述转化为使用重亚硫酸盐处理。Specifically, the subject is suspected of having cancer. Specifically, the sample collected from the subject is a plasma sample. Specifically, the conversion is treatment with bisulfite.
具体来说，所述探针组合物包括：靶向泛癌特异性区域的2个探针，靶向癌症特异性区域的n个探针，和靶向组织特异性区域的m个探针。所述探针组合物包括：低甲基化探针，其与经重亚硫酸盐转化的不含CG甲基化的所述癌症特异性区域、泛癌特异性区域、以及组织特异性区域杂交，和高甲基化探针，其与经重亚硫酸盐转化的CG全部甲基化的所述癌症特异性区域、泛癌特异性区域、以及组织特异性区域杂交。所述探针组合物中的每一个探针的长度为40～60bp。例如，所述探针组合物中的每一个探针的长度为45～56bp，优选50～56bp，进一步优选50bp。Specifically, the probe composition includes: 2 probes targeting a pan-cancer specific region, n probes targeting a cancer-specific region, and m probes targeting a tissue-specific region. The probe composition includes: a hypomethylation probe that hybridizes to the cancer-specific region, pan-cancer specific region, and tissue-specific region that are converted by bisulfite and do not contain CG methylation , And a hypermethylation probe that hybridizes to the cancer-specific region, pan-cancer-specific region, and tissue-specific region where the bisulfite-converted CG is fully methylated. The length of each probe in the probe composition is 40-60 bp. For example, the length of each probe in the probe composition is 45-56 bp, preferably 50-56 bp, and more preferably 50 bp.
具体来说，所述探针组合物中的n个探针靶向癌症特异性区域，其中，n为选自1-192中的任意的整数；其中，所述癌症特异性区域选自Seq ID No.：1-62中的任意。所述探针组合物中的m个探针靶向所述组织特异性区域，其中，m为选自1-116中的任意的整数；其中，所述组织特异性区域选自Seq ID No.：65-117中的任意。所述低甲基化探针包括靶向癌症特异性区域的探针Seq ID No.：118-214中的任意，靶向泛癌特异性区域的探针Seq ID No.：215-216中的任意，和靶向组织特异性区域的探针Seq ID No.：217-274中的任意。所述高甲基化探针包括靶向癌症特异性区域的探针Seq ID No.：275-371中的任意，靶向泛癌特异性区域的探针Seq ID No.：372-373中的任意，和靶向组织特异性区域的探针Seq ID No.：374-431中的任意。Specifically, the n probes in the probe composition target a cancer-specific region, where n is any integer selected from 1-192; wherein, the cancer-specific region is selected from Seq ID No.: Any of 1-62. The m probes in the probe composition target the tissue-specific region, where m is an integer selected from 1-116; wherein, the tissue-specific region is selected from Seq ID No. : Any of 65-117. The hypomethylation probes include probes targeting cancer-specific regions Seq ID No.: any of 118-214, and probes targeting pan-cancer specific regions Seq ID No.: 215-216 Any, and the probe Seq ID No. that targets tissue-specific regions: Any of 217-274. The hypermethylation probes include probes targeting cancer-specific regions, Seq ID No.: any of 275-371, and probes targeting pan-cancer specific regions, Seq ID No.: 372-373, Seq ID No.: any of 374-431 probes targeting tissue-specific regions.
具体来说，所述判读包括以下步骤：(1)比对泛癌特异性区域数据库，并进行判读以确认受试者是否患有癌症；(2)比对癌症特异性区域数据库，并进行判读以确认受试者患有的癌症为几种疑似癌症中的一种；(3)比对组织特异性区域数据库，并进行判读以确认受试者患癌的部位。所述步骤(1)包括进行如下判读：判断所述泛癌特异性区域Seq ID No.：63的甲基化水平是否大于等于55％，并且所述泛癌特异性区域Seq ID No.：64的甲基化水平是否大于等于60％，则判读所述患者患有癌症。所述步骤(2)包括进行如下判读：如果在靶向所述癌症特异性区域的n个探针中，n1个探针靶向的区域的甲基化水平大于等于各自阈值，且n1/n≥20％，优选n1/n≥30％，则判读患者患有组织特异性癌症中的任意一种。所述步骤(3)包括进行如下判读：在靶向所述组织特异性区域的m个探针中m1个探针靶向的区域的甲基化水平大于等于各自阈值，则进一步分析大于等于各自阈值的m1个探针所靶向的组织并计数每一个组织大于等于阈值的探针的个数，判读认为患者罹患癌症的组织是甲基化水平大于等于阈值的探针个数最多的组织。下表1中也给出了各靶区域的甲基化的阈值。Specifically, the interpretation includes the following steps: (1) compare the pan-cancer specific region database and perform interpretation to confirm whether the subject has cancer; (2) compare the cancer specific region database and perform interpretation To confirm that the subject's cancer is one of several suspected cancers; (3) Compare the tissue-specific area database and perform interpretation to confirm the subject's cancer site. The step (1) includes the following interpretation: judging whether the methylation level of the pan-cancer specific region Seq ID No.: 63 is greater than or equal to 55%, and the pan-cancer specific region Seq ID No.: 64 If the methylation level of is greater than or equal to 60%, it is judged that the patient has cancer. The step (2) includes the following interpretation: if among the n probes targeting the cancer-specific region, the methylation level of the region targeted by n1 probes is greater than or equal to the respective threshold, and n1/n ≥20%, preferably n1/n≥30%, then it is judged that the patient has any one of the tissue-specific cancers. The step (3) includes the following interpretation: among the m probes targeting the tissue-specific region, the methylation level of the region targeted by m1 probes is greater than or equal to the respective threshold, and then further analysis is greater than or equal to the respective threshold. Threshold m1 probes target tissues and count the number of probes in each tissue that are greater than or equal to the threshold, and judge that the tissue in which the patient suffers from cancer is the tissue with the largest number of probes with methylation level greater than or equal to the threshold. Table 1 below also shows the methylation threshold of each target region.
表1本申请中涉及的序列Table 1 Sequences involved in this application
Figure PCTCN2021081276-appb-000001
Figure PCTCN2021081276-appb-000001
Figure PCTCN2021081276-appb-000002
Figure PCTCN2021081276-appb-000002
Figure PCTCN2021081276-appb-000003
Figure PCTCN2021081276-appb-000003
Figure PCTCN2021081276-appb-000004
Figure PCTCN2021081276-appb-000004
Figure PCTCN2021081276-appb-000005
Figure PCTCN2021081276-appb-000005
Figure PCTCN2021081276-appb-000006
Figure PCTCN2021081276-appb-000006
Figure PCTCN2021081276-appb-000007
Figure PCTCN2021081276-appb-000007
Figure PCTCN2021081276-appb-000008
Figure PCTCN2021081276-appb-000008
Figure PCTCN2021081276-appb-000009
Figure PCTCN2021081276-appb-000009
Figure PCTCN2021081276-appb-000010
Figure PCTCN2021081276-appb-000010
Figure PCTCN2021081276-appb-000011
Figure PCTCN2021081276-appb-000011
Figure PCTCN2021081276-appb-000012
Figure PCTCN2021081276-appb-000012
Figure PCTCN2021081276-appb-000013
Figure PCTCN2021081276-appb-000013
Figure PCTCN2021081276-appb-000014
Figure PCTCN2021081276-appb-000014
Figure PCTCN2021081276-appb-000015
Figure PCTCN2021081276-appb-000015
Figure PCTCN2021081276-appb-000016
Figure PCTCN2021081276-appb-000016
Figure PCTCN2021081276-appb-000017
Figure PCTCN2021081276-appb-000017
Figure PCTCN2021081276-appb-000018
Figure PCTCN2021081276-appb-000018
实施例Example
实施例1：Example 1:
如图1所示，本申请的实施流程具体如下：As shown in Figure 1, the implementation process of this application is specifically as follows:
1.1.cfDNA提取纯化1.1. cfDNA extraction and purification
1.1.1.血浆样本制备：1.1.1. Plasma sample preparation:
4℃、2000g离心血液样本10min,将血浆转移到一个新的离心管中。4℃、16000g离心血浆样本10min,根据使用的收集管类型，执行下一步,本实验中使用的收集管类型为其他。Centrifuge the blood sample at 2000g for 10 minutes at 4°C, and transfer the plasma to a new centrifuge tube. Centrifuge plasma samples at 16000g for 10 minutes at 4°C. According to the type of collection tube used, proceed to the next step. The type of collection tube used in this experiment is other.
表2Table 2
Figure PCTCN2021081276-appb-000019
Figure PCTCN2021081276-appb-000019
1.1.2.裂解和结合1.1.2. Lysis and binding
1.1.2.1.按照下表准备结合溶液/珠子混合物，然后彻底混匀。1.1.2.1. Prepare the binding solution/bead mixture according to the following table, and then mix thoroughly.
表3table 3
Figure PCTCN2021081276-appb-000020
Figure PCTCN2021081276-appb-000020
加入适量体积的血浆样品。Add an appropriate volume of plasma sample.
1.1.2.2.彻底混匀血浆样品和结合溶液/珠子混合物。1.1.2.2. Thoroughly mix the plasma sample and the binding solution/bead mixture.
1.1.2.3.在旋转混匀仪上充分的结合10min，使cfDNA结合到磁珠上。1.1.2.3. Fully bind on the rotary mixer for 10 minutes to bind the cfDNA to the magnetic beads.
1.1.2.4.将结合管放在磁力架上5min，直到溶液变得澄清，磁珠完全吸附在磁力架上。1.1.2.4. Place the binding tube on the magnetic stand for 5 minutes until the solution becomes clear and the magnetic beads are completely adsorbed on the magnetic stand.
1.1.2.5.用移液管小心的弃去上清，继续保持管子在磁力架上几分钟，用移液管移去残留上清。1.1.2.5. Use a pipette to carefully discard the supernatant, keep the tube on the magnetic stand for a few minutes, and use a pipette to remove the remaining supernatant.
1.1.3.洗涤1.1.3. Washing
1.1.3.1.将珠子重悬在1ml洗涤溶液中。1.1.3.1. Resuspend the beads in 1ml washing solution.
1.1.3.2.将重悬液转移到新的无吸附1.5ml离心管中。保留结合管。1.1.3.2. Transfer the resuspension to a new non-adsorbent 1.5ml centrifuge tube. Keep the combined tube.
1.1.3.3.将含有珠子重悬液的离心管置于磁力架上，20s。1.1.3.3. Place the centrifuge tube containing the bead resuspension solution on the magnetic stand for 20 seconds.
1.1.3.4.将分离得到的上清，吸出洗涤结合管，将清洗后的残留珠子再次收集到重悬液中，弃掉裂解/结合管。1.1.3.4. Aspirate the separated supernatant and wash the binding tube, collect the washed residual beads into the resuspension solution again, and discard the lysis/binding tube.
1.1.3.5.管子置于磁力架上2min，直到溶液变得澄清，珠子聚集在磁力架，用1ml移液器移除上清。1.1.3.5. Place the tube on the magnetic stand for 2 minutes until the solution becomes clear and the beads gather on the magnetic stand. Use a 1ml pipette to remove the supernatant.
1.1.3.6.管子留在磁力架上，用200μL移液器尽可能移除残留的液体。1.1.3.6. Leave the tube on the magnetic stand, use a 200μL pipette to remove as much residual liquid as possible.
1.1.3.7.将管子从磁力架取下来，加入1ml洗涤溶液，涡旋30s。1.1.3.7. Remove the tube from the magnetic stand, add 1 ml of washing solution, and vortex for 30 seconds.
1.1.3.8.置于磁力架2min，直到溶液澄清，珠子聚集在磁力架上，用1ml移液管移除上清。1.1.3.8. Place on the magnetic stand for 2 minutes until the solution is clear and the beads gather on the magnetic stand. Remove the supernatant with a 1ml pipette.
1.1.3.9.管子留在磁力架上，用200μL移液器彻底移除残留液体。1.1.3.9. Leave the tube on the magnetic stand, and use a 200μL pipette to completely remove the remaining liquid.
1.1.3.10.将管子从磁力架取下，加入1ml 80％乙醇，涡旋30s。1.1.3.10. Remove the tube from the magnetic stand, add 1ml 80% ethanol, and vortex for 30s.
1.1.3.11.置于磁力架上2min，溶液变得澄清，用1ml移液器移去上清。1.1.3.11. Place on the magnetic stand for 2 minutes, the solution becomes clear, use a 1ml pipette to remove the supernatant.
1.1.3.12.管子留在磁力架上，用200μL移液器移去残留液体。1.1.3.12. The tube is left on the magnetic stand, and the remaining liquid is removed with a 200μL pipette.
1.1.3.13.用80％乙醇重复上述1.1.3.10.-1.1.3.12.步骤一次，尽可能除去上清。1.1.3.13. Repeat the above 1.1.3.10.-1.1.3.12. steps once with 80% ethanol, and remove the supernatant as much as possible.
1.1.3.14.管子留在磁力架上，空气中干燥珠子3～5分钟。1.1.3.14. Leave the tube on the magnetic stand, and dry the beads in the air for 3 to 5 minutes.
1.1.4.洗脱cfDNA1.1.4. Elution of cfDNA
1.1.4.1.按照下表加入洗脱液。1.1.4.1. Add the eluent according to the following table.
表4Table 4
Figure PCTCN2021081276-appb-000021
Figure PCTCN2021081276-appb-000021
Figure PCTCN2021081276-appb-000022
Figure PCTCN2021081276-appb-000022
1.1.4.2.涡旋5min，置于磁力架上2min，溶液变得澄清，吸取上清液中的cfDNA。1.1.4.2. Vortex for 5 minutes, place on the magnetic stand for 2 minutes, the solution becomes clear, absorb the cfDNA in the supernatant.
1.1.4.3.纯化的cfDNA立即使用，或者将上清转移至新的离心管中，-20℃保存。1.1.4.3. Use the purified cfDNA immediately, or transfer the supernatant to a new centrifuge tube and store at -20°C.
1.2.gDNA打断与纯化：1.2. gDNA interruption and purification:
1.2.1.按照Qubit浓度，取2μg gDNA，加水补至125μl，加入到covaris130μl打断管中，设置程序：50W，20％，200个循环，250s。1.2.1. According to Qubit concentration, take 2μg gDNA, add water to make up to 125μl, and add it to the 130μl interruption tube of covaris. Setting program: 50W, 20%, 200 cycles, 250s.
1.2.2.打断结束后取1μl样品使用Agilent2100进行片段检测，正常打断后样品检测主峰约在150bp-200bp。1.2.2. After the interruption, take 1μl of the sample and use the Agilent2100 for fragment detection. After the normal interruption, the main peak of the sample detection is about 150bp-200bp.
对于cfDNA样品，Agilent2100进行片段检测，直接Qubit用于后续的实验。For cfDNA samples, Agilent 2100 performs fragment detection, and Qubit is directly used in subsequent experiments.
1.3.末端修复、3‘端加“A”：1.3. End repair, 3'end with "A":
1.3.1.取20ng打断后的gDNA或cfDNA至PCR管中，用无核酸酶水补至50μl，加入以下试剂，涡旋混匀：1.3.1. Take 20ng of interrupted gDNA or cfDNA into a PCR tube, make up to 50μl with nuclease-free water, add the following reagents, and vortex to mix:
表5table 5
组分Component 体积volume
gDNA/cfDNAgDNA/cfDNA 50μl50μl
终止修复和A加尾缓冲液Termination of repair and A tailing buffer 7μl7μl
终止修复和A加尾酶混合物Termination Repair and A Tailing Enzyme Mix 3μl3μl
总体积total capacity 60μl60μl
1.3.2.设置以下程序在PCR仪上进行反应：热盖温度85℃。1.3.2. Set the following program to perform the reaction on the PCR machine: the temperature of the hot lid is 85°C.
表6Table 6
温度temperature 时间time
20℃20℃ 30min30min
65℃65°C 30min30min
4℃4℃ ∞∞
1.4.接头连接及纯化：1.4. Connector connection and purification:
1.4.1.参照下表将接头提前稀释成合适的浓度：1.4.1. Refer to the following table to dilute the joints in advance to an appropriate concentration:
表7Table 7
每50ul ER和AT反应的片段化的DNAFragmented DNA per 50ul ER and AT reaction 接头浓度Concentration of joint
1μg1μg 10uM10uM
500ng500ng 10uM10uM
250ng250ng 10uM10uM
100ng100ng 10uM10uM
50ng50ng 10uM10uM
25ng25ng 10uM10uM
10ng10ng 3uM3uM
5ng5ng 5uM5uM
2.5ng2.5ng 2.5uM2.5uM
1ng1ng 625nM625nM
1.4.2.按下表配制以下试剂，轻轻吸打混匀，短暂离心：1.4.2. Prepare the following reagents according to the table below, gently pipette to mix, and centrifuge briefly:
表8Table 8
组分Component 体积volume
末端修复、加“A”反应产物End repair, adding "A" reaction product 60μl60μl
接头Connector 5μl5μl
无核酸酶水Nuclease-free water 5μl5μl
连接缓冲液Connection buffer 30μl30μl
DNA连接酶DNA ligase 10μl10μl
总体积total capacity 110μl110μl
1.4.3.设置以下程序在PCR仪上进行反应：无热盖。1.4.3. Set up the following program to carry out the reaction on the PCR machine: no hot lid.
表9Table 9
温度temperature 时间time
20℃20℃ 30min30min
4℃4℃ ∞∞
1.4.4.按照以下体系，加入纯化磁珠进行实验(Agencourt AMPure XP磁珠提前拿至室温震荡混合均匀备用)：1.4.4. According to the following system, add purified magnetic beads for experiment (Agencourt AMPure XP magnetic beads are taken to room temperature in advance, shake and mix well for use):
表10Table 10
组分Component 体积volume
接头连接产物Connector connection product 110μl110μl
Agencourt AMPure XP珠子Agencourt AMPure XP beads 110μl110μl
总体积total capacity 220μl220μl
1.4.4.1.轻轻吸打混匀6次。1.4.4.1. Gently pipette and mix 6 times.
1.4.4.2.室温静置孵育5-15min，将PCR管置于磁力架上3min使溶液澄 清。1.4.4.2. Incubate at room temperature for 5-15 minutes. Place the PCR tube on the magnetic stand for 3 minutes to clear the solution.
1.4.4.3.移除上清，PCR管继续放置在磁力架上，向PCR管内加入200μl80％乙醇溶液，静置30s。1.4.4.3. Remove the supernatant, continue to place the PCR tube on the magnetic stand, add 200 μl of 80% ethanol solution to the PCR tube, and let it stand for 30 seconds.
1.4.4.4.移除上清，再向PCR管内加入200μl 80％乙醇溶液，静置30s后彻底移除上清(建议使用10μl移液器移除底部残留乙醇溶液)。1.4.4.4. Remove the supernatant, and then add 200μl 80% ethanol solution to the PCR tube, and let it stand for 30s to completely remove the supernatant (it is recommended to use a 10μl pipette to remove the remaining ethanol solution at the bottom).
1.4.4.5.室温静置3-5min，使残留乙醇彻底挥发。1.4.4.5. Let it stand at room temperature for 3-5 minutes to completely evaporate the residual ethanol.
1.4.4.6.加入22μl的无核酸酶水，把PCR管从磁力架取下，轻轻吸打重悬磁珠，避免产生气泡，室温静置2min。1.4.4.6. Add 22μl of nuclease-free water, remove the PCR tube from the magnetic stand, gently suck and resuspend the magnetic beads to avoid air bubbles, and let it stand at room temperature for 2 minutes.
1.4.4.7.将PCR管置于磁力架上2min使溶液澄清。1.4.4.7. Place the PCR tube on the magnetic stand for 2 minutes to clarify the solution.
1.4.4.8.用移液器吸取20μl上清液，转移到新的PCR管。1.4.4.8. Use a pipette to aspirate 20μl of supernatant and transfer to a new PCR tube.
1.5重亚硫酸盐处理及纯化：1.5 Bisulfite treatment and purification:
1.5.1.预先拿出所需要的试剂，并溶解。根据下表加入各试剂：1.5.1. Take out the required reagents in advance and dissolve them. Add the reagents according to the following table:
表11Table 11
组分Component 高浓度样品(1ng-2μg)体积High concentration sample (1ng-2μg) volume 低浓度样品(1-500ng)体积Low concentration sample (1-500ng) volume
接头连接纯化产物Adapter connects the purified product 20μl20μl 40μl40μl
重亚硫酸盐溶液Bisulfite solution 85μl85μl 85μl85μl
DNA保护缓冲液DNA protection buffer 35μl35μl 15μl15μl
总体积total capacity 140μl140μl 140μl140μl
1.5.2.DNA保护缓冲液加入液体变成蓝色。轻轻吸打混匀，然后分成两管至于PCR仪上。1.5.2. DNA protection buffer added to the liquid turns blue. Mix gently by pipetting, and then divide into two tubes on the PCR machine.
1.5.3.设置以下程序，并运行：热盖105℃。1.5.3. Set up the following program and run it: hot lid at 105°C.
表12Table 12
温度temperature 时间time
95℃95°C 5min5min
60℃60℃ 10min10min
95℃95°C 5min5min
60℃60℃ 10min10min
4℃4℃ ∞∞
1.5.4.简短离心将两管相同样本合并至同一个干净的1.5ml离心管中。1.5.4. Brief centrifugation to combine two tubes of the same sample into the same clean 1.5ml centrifuge tube.
1.5.5.每个样本中加入310μl缓冲液BL(样本量少于100ng加入1μl的载体RNA(1μg/μl))，涡旋混匀，简短离心。1.5.5. Add 310μl of buffer BL to each sample (add 1μl of carrier RNA (1μg/μl) for samples less than 100ng), vortex to mix, and centrifuge briefly.
1.5.6.加入250μl无水乙醇到每个样本中，涡旋混匀15s，简短离心，将混合液加入到准备好的对应的离心柱中。1.5.6. Add 250μl of absolute ethanol to each sample, vortex and mix for 15s, centrifuge briefly, and add the mixture to the prepared corresponding spin column.
1.5.7.静置1min，离心1min，将收集管中的液体重新转移到离心柱中， 离心1min，弃去离心管的液体。1.5.7. Let stand for 1 min, centrifuge for 1 min, transfer the liquid in the collection tube to the spin column again, centrifuge for 1 min, and discard the liquid in the centrifuge tube.
1.5.8.加入500μl缓冲液BW(注意是否加入无水乙醇)，离心1min，弃去废液。1.5.8. Add 500μl of buffer BW (note whether to add absolute ethanol), centrifuge for 1 min, and discard the waste solution.
1.5.9.加入500μl缓冲液BD(注意是否加入无水乙醇)，盖好管盖，室温放置15min。离心1min，弃去离心下的液体。1.5.9. Add 500μl of buffer BD (note whether to add absolute ethanol), cover the tube, and leave it at room temperature for 15 minutes. Centrifuge for 1 min and discard the liquid under the centrifugation.
1.5.10.加入500μl缓冲液BW(注意是否加入无水乙醇)，离心1min，弃去离下来的液体，在重复一次，共2次。1.5.10. Add 500μl of buffer BW (note whether to add absolute ethanol), centrifuge for 1 min, discard the separated liquid, repeat once for a total of 2 times.
1.5.11.加入250μl无水乙醇，离心1min，将离心柱放置到新的2ml收集管中，弃掉全部剩余液体。1.5.11. Add 250μl of absolute ethanol, centrifuge for 1min, place the spin column in a new 2ml collection tube, and discard all remaining liquid.
1.5.12.将离心柱放置到干净的1.5ml离心管中，加入20μl无核酸酶水到离心柱膜中心，轻轻盖上管盖，室温放置1min，离心1min。1.5.12. Place the spin column in a clean 1.5ml centrifuge tube, add 20μl of nuclease-free water to the center of the spin column membrane, cover the tube cap gently, leave it at room temperature for 1 min, and centrifuge for 1 min.
1.5.13.将收集管中的液体重新转移至离心柱中，室温放置1min，离心1min。1.5.13. Transfer the liquid in the collection tube to the spin column again, leave it at room temperature for 1 min, and centrifuge for 1 min.
1.6.杂交前预扩增及纯化：1.6. Pre-amplification and purification before hybridization:
1.6.1.按下列表格配制反应体系，吹打混匀，短暂离心：1.6.1. Prepare the reaction system according to the table below, mix by pipetting, and centrifuge briefly:
表13Table 13
Figure PCTCN2021081276-appb-000023
Figure PCTCN2021081276-appb-000023
1.6.2.设置以下程序并启动PCR程序：热盖105℃1.6.2. Set the following program and start the PCR program: hot lid 105℃
表14Table 14
Figure PCTCN2021081276-appb-000024
Figure PCTCN2021081276-appb-000024
1.6.3.PCR循环数根据投入DNA的量不同进行调整，参考数据如下所示：1.6.3. The number of PCR cycles is adjusted according to the amount of DNA input. The reference data is as follows:
表15Table 15
Figure PCTCN2021081276-appb-000025
Figure PCTCN2021081276-appb-000025
1.6.4.向反应结束后的PCR管中加入50μl Agencourt AMPure XP磁珠，用移液器吹打混匀，避免产生气泡(Agencourt AMPure XP提前室温混匀并平衡)。1.6.4. Add 50μl Agencourt AMPure XP magnetic beads to the PCR tube after the reaction, pipette and mix to avoid air bubbles (Agencourt AMPure XP mix and balance at room temperature in advance).
1.6.5.室温孵育5-15min，把PCR管置于磁力架上3min使溶液澄清。1.6.5. Incubate at room temperature for 5-15 minutes, and place the PCR tube on the magnetic stand for 3 minutes to clarify the solution.
1.6.6.移除上清，PCR管继续放置在磁力架上，向PCR管内加入200μl80％乙醇溶液，静置30s。1.6.6. Remove the supernatant, continue to place the PCR tube on the magnetic stand, add 200μl of 80% ethanol solution to the PCR tube, and let it stand for 30s.
1.6.7.移除上清，再向PCR管内加入200μl 80％乙醇溶液，静置30s后彻底移除上清(建议使用10μl移液器移除底部残留乙醇溶液)。1.6.7. Remove the supernatant, and then add 200μl 80% ethanol solution to the PCR tube, let it stand for 30s and then completely remove the supernatant (it is recommended to use a 10μl pipette to remove the remaining ethanol solution at the bottom).
1.6.8.室温静置5min，使残留乙醇彻底挥发。1.6.8. Leave it at room temperature for 5 minutes to completely evaporate the residual ethanol.
1.6.9.加入30μl的无核酸酶水，将离心管从磁力架取下，使用移液器，轻轻吸打重悬磁珠。1.6.9. Add 30μl of nuclease-free water, remove the centrifuge tube from the magnetic stand, and use a pipette to gently pipette and resuspend the magnetic beads.
1.6.10.室温静置2min，将200μl PCR管置于磁力架上2min使溶液澄清。1.6.10. Let it stand at room temperature for 2 minutes, and place the 200μl PCR tube on the magnetic stand for 2 minutes to clarify the solution.
1.6.11.用移液器将上清液转移到新的200μl PCR管中(置于冰盒上)，在反应管上标记好样本号，准备下一步反应。1.6.11. Use a pipette to transfer the supernatant to a new 200μl PCR tube (placed on the ice box), mark the sample number on the reaction tube, and prepare for the next reaction.
1.6.12.取1μl样品使用Qubit进行文库浓度测定，记录文库浓度。1.6.12. Take 1μl sample and use Qubit to determine the library concentration, and record the library concentration.
1.6.13.取1μl样品使用安捷伦2100进行文库片段长度测定，文库长度约在270bp-320bp间。1.6.13. Take 1μl sample and use Agilent 2100 to determine the length of library fragments. The length of the library is about 270bp-320bp.
1.7.样品与探针杂交：1.7. Hybridization of sample and probe:
1.7.1.按照以下体系将样品文库与各种Hyb阻断物混匀，标记为B：1.7.1. Mix the sample library with various Hyb blockers according to the following system and mark it as B:
表16Table 16
组分Component 体积volume
预扩增产物Pre-amplified product 750ng对应体积750ng corresponding volume
Hyb人阻断物Hyb human blocker 5μl5μl
接头阻断物Junction blocker 6μl6μl
增强剂Enhancer 5μl5μl
1.7.2.将准备好的样品和Hyb阻断物混合物放入真空浓缩离心机，打开PCR管盖，启动离心机，打开真空泵开关，开始浓缩。1.7.2. Put the prepared sample and Hyb blocker mixture into the vacuum concentration centrifuge, open the PCR tube cover, start the centrifuge, turn on the vacuum pump switch, and start concentration.
1.7.3.将抽干的样品重新溶在约9μl无核酸酶水中,总体积10μl，轻轻吸打混匀，短暂离心后置于冰上待用，标记为B。1.7.3. Re-dissolve the drained sample in about 9μl of nuclease-free water, with a total volume of 10μl, gently pipette to mix, centrifuge briefly and place on ice for later use, marked as B.
1.7.4.将Hyb缓冲液置于室温融化，融解之后会有沉淀出现，混匀后置于65℃水浴锅内预热，完全溶解后(无沉淀及浑浊物)取20μl Hyb缓冲液置于新的200μl PCR管内，盖好管盖,标记为A,继续置于65℃水浴锅内孵育待用。1.7.4. Put the Hyb buffer solution at room temperature to melt, there will be precipitation after melting, and place it in a 65℃ water bath to preheat it after mixing. After completely dissolved (no precipitation and turbidity), take 20μl Hyb buffer solution and place In the new 200μl PCR tube, close the tube cap, mark it as A, and continue to incubate it in a 65℃ water bath for later use.
1.7.5.通过艾吉泰康生物科技(北京)有限公司合成以下低甲基化探针：1.7.5. Synthesize the following hypomethylation probes through Aiji Taikang Biotechnology (Beijing) Co., Ltd.:
a)靶向癌症特异性区域的探针Seq ID No.：118-214中的任意，b)靶向泛癌特异性区域的探针Seq ID No.：215-216中的任意，和c)靶向组织特异性区域的探针Seq ID No.：217-274中的任意，a) Seq ID No. of probes targeting cancer-specific regions: any of 118-214, b) Seq ID No. of probes targeting cancer-specific regions: any of 215-216, and c) Seq ID No.: any of 217-274 probes targeting tissue-specific regions,
并且合成以下高甲基化探针：And synthesize the following hypermethylation probes:
d)靶向癌症特异性区域的探针Seq ID No.：275-371中的任意，e)靶向泛癌特异性区域的探针Seq ID No.：372-373中的任意，和f)靶向组织特异性区域的探针Seq ID No.：374-431中的任意。d) Seq ID No. of probes targeting cancer-specific regions: any of 275-371, e) Seq ID No. of probes targeting cancer-specific regions: any of 372-373, and f) Seq ID No.: Any of 374-431 probes targeting tissue-specific regions.
并且以a:b:c:d:e:f＝1:1:1:1:1:1的比例制成探针组合物。And the probe composition is made with a ratio of a:b:c:d:e:f=1:1:1:1:1:1.
1.7.6.取5μl RNA酶阻断物与2μl探针组合物置于200μl PCR管内，轻轻吸打混匀，短暂离心后置于冰上待用，标记为C。1.7.6. Take 5μl of RNase blocker and 2μl of the probe composition into a 200μl PCR tube, gently pipette to mix, centrifuge briefly and place on ice for later use, labeled C.
1.7.7.设置PCR仪参数，热盖100℃，95℃，5min；65℃，保持。1.7.7. Set the PCR instrument parameters, heat the lid to 100°C, 95°C, 5min; 65°C, keep it.
1.7.8.将PCR管B置于PCR仪上，运行以上程序。1.7.8. Place PCR tube B on the PCR machine and run the above procedure.
1.7.9.PCR仪温度降至65℃时，将PCR管A置于PCR仪上孵育，盖上PCR仪热盖。1.7.9. When the temperature of the PCR instrument drops to 65°C, place the PCR tube A on the PCR instrument and incubate, and cover the thermal lid of the PCR instrument.
1.7.10.5min后，将C置于PCR上孵育，盖上PCR仪热盖。1.7.1 After 0.5 min, place C on the PCR and incubate, and cover the thermal lid of the PCR machine.
1.7.11.将PCR管C放置入PCR仪2min后，把移液器调至13μl，从PCR管A中吸取13μl Hyb缓冲液移至PCR管C中，吸取全部PCR管B中样品移至PCR管C中，轻轻吸打10次，充分混匀，避免产生大量气泡，密封管盖，盖上PCR仪热盖,65℃孵育过夜(16-24h)。1.7.11. After placing PCR tube C in the PCR machine for 2 minutes, adjust the pipette to 13μl, draw 13μl Hyb buffer from PCR tube A and transfer it to PCR tube C, draw all the samples in PCR tube B and transfer it to PCR In tube C, gently pipette 10 times and mix thoroughly to avoid a large number of bubbles. Seal the tube cap, cover the thermal lid of the PCR machine, and incubate at 65°C overnight (16-24h).
1.8.捕获目标区域DNA文库：1.8. Capture the DNA library of the target region:
1.8.1.捕获磁珠的准备1.8.1. Preparation of capture magnetic beads
1.8.1.1.将磁珠(Dynabeads MyOne Streptavidin T1磁珠)从4℃取出，涡旋震荡重悬。1.8.1.1. Take out the magnetic beads (Dynabeads MyOne Streptavidin T1 magnetic beads) from 4°C, vortex and resuspend.
1.8.1.2.取50μl磁珠置于新的PCR管内，置于磁力架上1min使溶液澄清，移除上清。1.8.1.2. Take 50μl magnetic beads and place them in a new PCR tube, place them on the magnetic stand for 1 min to clarify the solution, and remove the supernatant.
1.8.1.3.从磁力架上取下PCR管，加入200μL结合缓冲液轻轻吸打数次混匀，重悬磁珠。1.8.1.3. Remove the PCR tube from the magnetic stand, add 200μL of binding buffer and gently pipette several times to mix, and resuspend the magnetic beads.
1.8.1.4.置磁力架上1min，移除上清。1.8.1.4. Put on the magnetic stand for 1 min, and remove the supernatant.
1.8.1.5.重复步骤3-4两次，共清洗磁珠3次。1.8.1.5. Repeat steps 3-4 twice to clean the magnetic beads 3 times.
1.8.1.6.从磁力架上取下PCR管，加入200μL结合缓冲液轻轻吸打6次重悬磁珠待用。1.8.1.6. Remove the PCR tube from the magnetic stand, add 200μL of binding buffer and gently pipette 6 times to resuspend the magnetic beads for later use.
1.8.2.捕获目标DNA文库1.8.2. Capture target DNA library
1.8.2.1.保持杂交产物PCR管C在PCR仪上，将准备好的200μL捕获磁珠加入到杂交后的产物PCR管C中，用移液器吸打6次混匀，置于旋转混匀仪上室温结合30min(转速最好不要超过10转/min)。1.8.2.1. Keep the hybridization product PCR tube C on the PCR machine, add 200μL of the prepared capture magnetic beads to the hybridization product PCR tube C, pipette 6 times to mix, and place it on the rotary mixer. Combine room temperature on the instrument for 30 min (rotation speed should not exceed 10 revolutions/min).
1.8.2.2.将PCR管置于磁力架上2min使溶液澄清，移除上清液。1.8.2.2. Place the PCR tube on the magnetic stand for 2 minutes to clarify the solution, and remove the supernatant.
1.8.2.3.向PCR管C内加入200μL的洗涤缓冲液1，轻轻吸打6次混匀，置于旋转混匀仪上清洗15min(转速最好不要超过10转/min)，然后短暂离心，将PCR管放于磁力架上2min使溶液澄清，移除上清。1.8.2.3. Add 200μL of washing buffer 1 to PCR tube C, gently pipette 6 times to mix, and place on a rotary mixer to wash for 15 minutes (the rotation speed should not exceed 10 revolutions/min), and then centrifuge briefly , Put the PCR tube on the magnetic stand for 2 minutes to clarify the solution, and remove the supernatant.
1.8.2.4.加入200μl的65℃预热后的洗涤缓冲液2，轻轻吸打6次混匀，置于混匀仪上65℃孵育10min，转速800转/min进行清洗。1.8.2.4. Add 200μl of washing buffer 2 preheated at 65°C, gently pipette 6 times to mix, and incubate on a mixer at 65°C for 10 minutes at a rotation speed of 800 rpm for washing.
1.8.2.5.短暂离心，将PCR管放于磁力架上2min，移除上清。使用洗涤缓冲液2再重复2次清洗，共计3次。最后一次彻底移除洗涤缓冲液2。1.8.2.5. Centrifuge briefly, put the PCR tube on the magnetic stand for 2 minutes, and remove the supernatant. Use Wash Buffer 2 to repeat the washing 2 more times for a total of 3 times. Remove Wash Buffer 2 thoroughly for the last time.
1.8.2.6.PCR管继续置于磁力架上，向PCR管内加入200μl 80％乙醇，静置30s后彻底移除乙醇溶液，室温晾干2min。1.8.2.6. Continue to place the PCR tube on the magnetic stand, add 200μl 80% ethanol to the PCR tube, completely remove the ethanol solution after standing for 30s, and dry at room temperature for 2min.
1.8.2.7.向PCR管加入30μL无核酸酶水，从磁力架上取下PCR管，轻轻吸打6次重悬磁珠待用。1.8.2.7. Add 30μL of nuclease-free water to the PCR tube, remove the PCR tube from the magnetic stand, and gently pipette 6 times to resuspend the magnetic beads for later use.
1.9.捕获后扩增及纯化1.9. Amplification and purification after capture
1.9.1.根据下表配制反应体系进行捕获文库的富集，轻轻吹打混匀后，短暂离心：1.9.1. Prepare a reaction system according to the following table to enrich the capture library, gently pipette to mix, and centrifuge briefly:
表17Table 17
Figure PCTCN2021081276-appb-000026
Figure PCTCN2021081276-appb-000026
1.9.2.设置以下程序，将样品置于PCR仪中，运行程序：热盖105℃。1.9.2. Set up the following program, put the sample in the PCR machine, and run the program: heat lid at 105°C.
表18Table 18
Figure PCTCN2021081276-appb-000027
Figure PCTCN2021081276-appb-000027
1.9.3.PCR结束后向样品加入55μl Agencourt AMPure XP磁珠，用移液器轻轻吸打混匀。1.9.3. After PCR, add 55μl Agencourt AMPure XP magnetic beads to the sample, and gently pipette to mix.
1.9.4.室温孵育5min，把PCR管置于磁力架上3min使溶液澄清。1.9.4. Incubate for 5 minutes at room temperature, and place the PCR tube on the magnetic stand for 3 minutes to clarify the solution.
1.9.5.移除上清，PCR管继续置于磁力架上，加入200μl 80％无水乙醇，静置30s。1.9.5. Remove the supernatant, continue to place the PCR tube on the magnetic stand, add 200μl 80% absolute ethanol, and let it stand for 30s.
1.9.6.移除上清，再向PCR管内加入200μl 80％无水乙醇，静置30后彻底移除上清。1.9.6. Remove the supernatant, and then add 200μl 80% absolute ethanol to the PCR tube, and let it stand for 30 to completely remove the supernatant.
1.9.7.室温放置5min，使得残留乙醇彻底挥发。1.9.7. Leave it at room temperature for 5 minutes to allow the residual ethanol to evaporate completely.
1.9.8.加入25μl无核酸酶水，将PCR管从磁力架拿下，轻轻吹打混匀重悬磁珠，室温放置2min。1.9.8. Add 25μl of nuclease-free water, remove the PCR tube from the magnetic stand, gently pipette to mix and resuspend the magnetic beads, and place at room temperature for 2 minutes.
1.9.9.将PCR管置于磁力架上2min使溶液澄清。1.9.9. Place the PCR tube on the magnetic stand for 2 minutes to clarify the solution.
1.9.10.用移液器吸23μl上清液转移到1.5ml离心管，标记样品信息。1.9.10. Use a pipette to aspirate 23μl of supernatant and transfer it to a 1.5ml centrifuge tube, and mark the sample information.
1.9.11.取1μl文库使用Qubit进行定量，记录文库浓度。1.9.11. Take 1μl of the library and use Qubit for quantification, and record the library concentration.
1.9.12.取1μl样品使用Agilent2100进行文库片段长度测定。1.9.12. Take 1μl sample and use Agilent2100 to measure the length of library fragments.
1.9.13.使用Illumina高通量测序平台进行测序。1.9.13. Use the Illumina high-throughput sequencing platform for sequencing.
1.10.甲基化生信分析流程。大致如下：使用trimmomatic等质控软件查看测序质量，去除低质量的读段，然后采用Bismarker等比对软件将质控后的干净的数据比对到参考基因组上，采用methykit等R包提取相应的甲基化位点。最后，计算出Panel上的每个靶区域的甲基化比率。1.10. Methylation bio-information analysis process. It is roughly as follows: Use quality control software such as trimmomatic to check the quality of sequencing, remove low-quality reads, and then use comparison software such as Bismarker to compare the clean data after quality control to the reference genome, and use R packages such as methykit to extract the corresponding Methylation site. Finally, calculate the methylation ratio of each target area on the Panel.
实施例2Example 2
一例经病理鉴定的胃癌样本，采用本申请的Panel检测，按实施例1的方法采集外周血；建库，并通过Illumina平台测序；测序数据经上述生物信息的分析流程，得到甲基化水平，结果如下表19所示(表19显示了检测到的大于等于甲基化阈值的靶区域)。A sample of gastric cancer identified by pathology was detected by the Panel of this application, and peripheral blood was collected according to the method of Example 1; the database was constructed and sequenced on the Illumina platform; the sequencing data was subjected to the above-mentioned biological information analysis process to obtain the methylation level, The results are shown in Table 19 below (Table 19 shows the detected target regions greater than or equal to the methylation threshold).
表19Table 19
基因Gene CHRCHR 起始Start 终止termination 甲基化比率Methylation ratio 靶区域序列号Target area serial number
TBX15TBX15 11 119527108119527108 119527157119527157 0.550.55 Seq ID No.63Seq ID No. 63
CRYGDCRYGD 22 208989200208989200 208989249208989249 0.600.60 Seq ID No.64Seq ID No. 64
CPECPE 44 166300051166300051 166300291166300291 0.420.42 Seq ID No.81Seq ID No.81
CPECPE 44 166300242166300242 166300291166300291 0.420.42 Seq ID No.82Seq ID No. 82
PLXDC2PLXDC2 1010 2010449720104497 2010454620104546 0.460.46 Seq ID No.97Seq ID No. 97
PLXDC2PLXDC2 1010 2010475820104758 2010480720104807 0.460.46 Seq ID No.98Seq ID No. 98
PLXDC2PLXDC2 1010 2010494820104948 2010499720104997 0.530.53 Seq ID No.99Seq ID No.99
PLXDC2PLXDC2 1010 2010559320105593 2010564220105642 0.490.49 Seq ID No.100Seq ID No. 100
OTX1OTX1 22 6328113963281139 6328118863281188 0.570.57 Seq ID No.11Seq ID No.11
SFRP2SFRP2 44 154710475154710475 154710536154710536 0.400.40 Seq ID No.19Seq ID No.19
SFRP2SFRP2 44 154710598154710598 154710647154710647 0.370.37 Seq ID No.20Seq ID No. 20
SFRP2SFRP2 44 154710702154710702 154710751154710751 0.360.36 Seq ID No.21Seq ID No. 21
SFRP2SFRP2 44 154710796154710796 154710845154710845 0.430.43 Seq ID No.22Seq ID No. 22
CDO1CDO1 55 115152372115152372 115152432115152432 0.480.48 Seq ID No.24Seq ID No. 24
CDO1CDO1 55 115152485115152485 115152543115152543 0.480.48 Seq ID No.25Seq ID No. 25
TRIM15TRIM15 66 3013100130131001 3013105030131050 0.570.57 Seq ID No.84Seq ID No. 84
TRIM15TRIM15 66 3013170130131701 3013176830131768 0.620.62 Seq ID No.31Seq ID No. 31
ALX4ALX4 1111 4433090344330903 4433095244330952 0.490.49 Seq ID No.47Seq ID No. 47
ALX4ALX4 1111 4433095844330958 4433100744331007 0.370.37 Seq ID No.48Seq ID No. 48
CCNA1CCNA1 1313 3700455337004553 3700461837004618 0.420.42 Seq ID No.53Seq ID No. 53
CCNA1CCNA1 1313 3700462037004620 3700466937004669 0.470.47 Seq ID No.54Seq ID No. 54
CCNA1CCNA1 1313 3700544137005441 3700550237005502 0.440.44 Seq ID No.55Seq ID No. 55
CCNA1CCNA1 1313 3700556637005566 3700563137005631 0.390.39 Seq ID No.56Seq ID No. 56
对检测样本进行模式识别分类鉴定，首先判读出泛癌特异性标志物TBX15和CRYGD基因的甲基化水平大于等于55％和60％，则初步判断该 样本为患有癌症的样本；其次，判读出癌症特异性标志物OTX1、SFRP2、CDO1、TRIM15、ALX4、CCNA1的甲基化水平均大于等于表1中所示的各自阈值(如上表19所示)，则进一步判断该样本为患有以下11种癌症(食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌)中的任意癌症的样本；最后，判读组织特异性标志物，基于表19中大于等于各自阈值的靶区域，可以看出属于胃组织特异性的6个靶区域Seq ID No.81、Seq ID No.82、Seq ID No.97、Seq ID No.98、Seq ID No.99和Seq ID No.100大于等于各自的甲基化水平的阈值，没有其他的组织特异性标志物的甲基化大于等于其各自的阈值，因此在大于等于各自甲基化阈值的组织特异性标志物中，胃组织特异性标志物最多，则最终判断该样本为患有胃癌的样本。Perform pattern recognition classification and identification of the test sample. Firstly, determine the methylation levels of pan-cancer specific markers TBX15 and CRYGD genes greater than or equal to 55% and 60%, and then preliminarily determine that the sample is a sample suffering from cancer; The methylation levels of cancer-specific markers OTX1, SFRP2, CDO1, TRIM15, ALX4, and CCNA1 are greater than or equal to the respective thresholds shown in Table 1 (as shown in Table 19 above), and then the sample is further judged as suffering from the following 11 types A sample of any cancer (esophageal cancer, stomach cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer); finally, interpret tissue-specific markers Based on the target regions that are greater than or equal to their respective thresholds in Table 19, it can be seen that there are 6 target regions specific to gastric tissue Seq ID No. 81, Seq ID No. 82, Seq ID No. 97, Seq ID No. 98, Seq ID No. 99 and Seq ID No. 100 are greater than or equal to their respective thresholds of methylation levels, and no other tissue-specific markers have methylation greater than or equal to their respective thresholds, so they are greater than or equal to their respective methylation thresholds Among the tissue-specific markers, gastric tissue-specific markers are the most, and the sample is finally judged to be a sample with gastric cancer.
该患者术后48小时，再次抽血，采用本申请的Panel检测，按实施例1的方法采集外周血；建库，并通过Illumina平台测序；测序数据经上述生物信息的分析流程，通过模式识别的方法，分析所有测序数据，结果显示上表中的基因甲基化水平回归正常水平。The patient’s blood was drawn again 48 hours after the operation, and the peripheral blood was collected using the Panel test of the application according to the method of Example 1. The database was constructed and sequenced on the Illumina platform; the sequencing data was subjected to the above-mentioned biological information analysis process and passed pattern recognition By analyzing all the sequencing data, the results show that the gene methylation level in the above table has returned to the normal level.
实施例3Example 3
一例结直肠癌样本，采用本申请的Panel检测，按实施例1的方法采集外周血；建库，并通过Illumina平台测序；测序数据经上述生物信息的分析流程，得到甲基化水平，结果如下表20所示(表20显示了检测到的大于等于甲基化阈值的靶区域)。A sample of colorectal cancer was detected by the Panel of this application, and peripheral blood was collected according to the method of Example 1; the database was constructed and sequenced on the Illumina platform; the sequencing data was subjected to the above-mentioned biological information analysis process to obtain the methylation level, and the results are as follows This is shown in Table 20 (Table 20 shows the detected target regions greater than or equal to the methylation threshold).
表20Table 20
基因Gene CHRCHR 起始Start 终止termination 甲基化比率Methylation ratio 靶区域序列号Target area serial number
TBX15TBX15 11 119527108119527108 119527157119527157 0.550.55 Seq ID No.63Seq ID No. 63
CRYGDCRYGD 22 208989200208989200 208989249208989249 0.600.60 Seq ID No.64Seq ID No. 64
C6orf155C6orf155 66 7213035972130359 7213040872130408 0.560.56 Seq ID No.87Seq ID No.87
C6orf155C6orf155 66 7213055372130553 7213060272130602 0.710.71 Seq ID No.88Seq ID No. 88
C6orf155C6orf155 66 7213064172130641 7213069072130690 0.650.65 Seq ID No.89Seq ID No. 89
C6orf155C6orf155 66 7213075572130755 7213080472130804 0.690.69 Seq ID No.90Seq ID No. 90
SHISA2SHISA2 1313 2662527326625273 2662539726625397 0.610.61 Seq ID No.110Seq ID No.110
TRHTRH 33 129693370129693370 129693434129693434 0.660.66 Seq ID No.16Seq ID No. 16
TRHTRH 33 129693586129693586 129693662129693662 0.530.53 Seq ID No.17Seq ID No. 17
CDO1CDO1 55 115152372115152372 115152432115152432 0.480.48 Seq ID No.24Seq ID No. 24
CDO1CDO1 55 115152485115152485 115152543115152543 0.480.48 Seq ID No.25Seq ID No. 25
ELMO1ELMO1 77 3748851637488516 3748857837488578 0.40.4 Seq ID No.35Seq ID No. 35
GFRA1GFRA1 1010 118032831118032831 118032906118032906 0.330.33 Seq ID No.40Seq ID No. 40
GFRA1GFRA1 1010 118032948118032948 118032997118032997 0.520.52 Seq ID No.41Seq ID No. 41
CCNA1CCNA1 1313 3700455337004553 3700461837004618 0.420.42 Seq ID No.53Seq ID No. 53
CCNA1CCNA1 1313 3700462037004620 3700466937004669 0.450.45 Seq ID No.54Seq ID No. 54
CCNA1CCNA1 1313 3700544137005441 3700550237005502 0.390.39 Seq ID No.55Seq ID No. 55
CCNA1CCNA1 1313 3700556637005566 3700563137005631 0.330.33 Seq ID No.56Seq ID No. 56
SALL1SALL1 1616 5118437951184379 5118444151184441 0.630.63 Seq ID No.58Seq ID No. 58
对检测样本进行模式识别分类鉴定，首先判读出泛癌特异性标志物TBX15和CRYGD基因的甲基化水平大于等于55％和60％，则初步判断该样本为患有癌症的样本；其次，判读出癌症特异性标志物TRH、CDO1、ELMO1、GFRA1、CCNA1、SALL1的甲基化水平均大于等于表1中所示的各自阈值(如上表20所示)，则进一步判断该样本为患有以下11种癌症(食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌)中的任意癌症的样本；最后，判读组织特异性标志物，基于表20中大于等于各自阈值的靶区域，可以看出属于结直肠组织特异性的6个靶区域Seq ID No.63、Seq ID No.64、Seq ID No.87、Seq ID No.88、Seq ID No.89、Seq ID No.90大于等于各自的甲基化水平的阈值，没有其他的组织特异性标志物的甲基化大于等于其各自的阈值，因此在大于等于各自甲基化阈值的组织特异性标志物中，结直肠组织特异性标志物最多，则最终判断该样本为患有结直肠癌的样本。Perform pattern recognition classification and identification of the test sample. Firstly, determine the methylation levels of pan-cancer specific markers TBX15 and CRYGD genes greater than or equal to 55% and 60%, and then preliminarily determine that the sample is a sample suffering from cancer; The methylation levels of the cancer-specific markers TRH, CDO1, ELMO1, GFRA1, CCNA1, and SALL1 are greater than or equal to the respective thresholds shown in Table 1 (as shown in Table 20 above), and then the sample is further judged as suffering from the following 11 types A sample of any cancer (esophageal cancer, stomach cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer); finally, interpret tissue-specific markers Based on the target regions in Table 20 that are greater than or equal to their respective thresholds, it can be seen that there are 6 target regions specific to colorectal tissues Seq ID No. 63, Seq ID No. 64, Seq ID No. 87, Seq ID No. 88 , Seq ID No. 89, Seq ID No. 90 are greater than or equal to their respective thresholds of methylation levels, and no other tissue-specific markers have methylation greater than or equal to their respective thresholds, so they are greater than or equal to their respective methylation thresholds. Among the threshold tissue-specific markers, colorectal tissue-specific markers are the most, and the sample is finally judged to be a sample with colorectal cancer.
患者术后48小时，再次抽血，采用本申请的Panel检测，按实施例1的方法采集外周血，建库，测序；测序数据经上述生物信息的分析流程，通过模式识别的方法，分析所有测序数据，结果显示上表中的基因甲基化水平回归正常水平。After 48 hours after the operation of the patient, the blood was drawn again, the Panel test of this application was used, the peripheral blood was collected according to the method of Example 1, the database was built, and the sequence was sequenced; the sequencing data was analyzed through the above-mentioned biological information analysis process and through the method of pattern recognition. Sequencing data shows that the gene methylation levels in the table above have returned to normal levels.
实施例4Example 4
一例肺癌样本，采用本申请的Panel检测，按实施例1的方法采集外周血；建库，并通过Illumina平台测序；测序数据经上述生物信息的分析流程，得到甲基化水平，结果如下表21所示(表21显示了检测到的大于等于甲基化阈值的靶区域)。A lung cancer sample was detected by the Panel of this application, and peripheral blood was collected according to the method in Example 1; the database was constructed and sequenced on the Illumina platform; the sequencing data was subjected to the above-mentioned biological information analysis process to obtain the methylation level. The results are shown in Table 21 As shown (Table 21 shows the detected target regions greater than or equal to the methylation threshold).
表21Table 21
基因Gene CHRCHR 起始Start 终止termination 甲基化比率Methylation ratio 靶区域序列号Target area serial number
TBX15TBX15 11 119527108119527108 119527157119527157 0.550.55 Seq ID No.63Seq ID No. 63
CRYGDCRYGD 22 208989200208989200 208989249208989249 0.600.60 Seq ID No.64Seq ID No. 64
FMO3FMO3 11 171059887171059887 171059936171059936 0.330.33 Seq ID No.71Seq ID No. 71
FMO3FMO3 11 171060010171060010 171060059171060059 0.330.33 Seq ID No.72Seq ID No. 72
ITIH3ITIH3 33 5282874652828746 5282881952828819 0.250.25 Seq ID No.76Seq ID No. 76
CHST12CHST12 77 24735292473529 24735782473578 0.510.51 Seq ID No.91Seq ID No.91
CHST12CHST12 77 24738112473811 24738602473860 0.510.51 Seq ID No.92Seq ID No. 92
RUNX3RUNX3 11 2525745725257457 2525758825257588 0.600.60 Seq ID No.1Seq ID No. 1
RUNX3RUNX3 11 2525813325258133 2525819525258195 0.570.57 Seq ID No.2Seq ID No. 2
RUNX3RUNX3 11 2525822525258225 2525828525258285 0.520.52 Seq ID No.3Seq ID No. 3
APCAPC 55 112073300112073300 112073439112073439 0.390.39 Seq ID No.23Seq ID No. 23
HOXA11ASHOXA11AS 77 2722542827225428 2722549727225497 0.40.4 Seq ID No.33Seq ID No. 33
HOXA11ASHOXA11AS 77 2722552327225523 2722557727225577 0.470.47 Seq ID No.34Seq ID No. 34
PHOX2APHOX2A 1111 7195493471954934 7195498371954983 0.530.53 Seq ID No.49Seq ID No. 49
GALR1GALR1 1818 7496191874961918 7496200174962001 0.490.49 Seq ID No.61Seq ID No. 61
对检测样本进行模式识别分类鉴定，首先判读出泛癌特异性标志物TBX15和CRYGD基因的甲基化水平大于等于55％和60％，则初步判断该样本为患有癌症的样本；其次，判读出癌症特异性标志物RUNX3、APC、HOXA11AS、PHOX2A、GALR1的甲基化水平均大于等于表1中所示的各自阈值(如上表21所示)，则进一步判断该样本为患有以下11种癌症(食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌)中的任意癌症的样本；最后，判读组织特异性标志物，基于表21中大于等于各自阈值的靶区域，可以看出属于肺组织特异性的5个靶区域Seq ID No.71、Seq ID No.72、Seq ID No.76、Seq ID No.91和Seq ID No.92大于等于各自的甲基化水平的阈值，没有其他的组织特异性标志物的甲基化大于等于其各自的阈值，因此在大于等于各自甲基化阈值的组织特异性标志物中，肺组织特异性标志物最多，则最终判断该样本为患有肺癌的样本。Perform pattern recognition classification and identification of the test sample. Firstly, determine the methylation levels of pan-cancer specific markers TBX15 and CRYGD genes greater than or equal to 55% and 60%, and then preliminarily determine that the sample is a sample suffering from cancer; The methylation levels of cancer-specific markers RUNX3, APC, HOXA11AS, PHOX2A, and GALR1 are greater than or equal to the respective thresholds shown in Table 1 (as shown in Table 21 above), and the sample is further judged to have the following 11 cancers ( Esophageal cancer, stomach cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and endometrial cancer) samples of any cancer; finally, the interpretation of tissue-specific markers, based on In Table 21, the target regions that are greater than or equal to their respective thresholds can be seen as five target regions Seq ID No. 71, Seq ID No. 72, Seq ID No. 76, Seq ID No. 91, and Seq ID that are specific to lung tissue No. 92 is greater than or equal to the respective threshold of methylation level, and no other tissue-specific markers have methylation greater than or equal to their respective thresholds. Therefore, in the tissue-specific markers greater than or equal to their respective methylation thresholds, If the lung tissue has the most specific markers, the sample is finally judged to be a sample with lung cancer.
患者术后48小时，再次抽血，采用本申请的Panel检测，按实施例1的方法采集外周血，建库，测序；测序数据经上述生物信息的分析流程，通过模式识别的方法，分析所有测序数据，结果显示上表中的基因甲基化水平回归正常水平。After 48 hours after the operation of the patient, the blood was drawn again, the Panel test of this application was used, the peripheral blood was collected according to the method of Example 1, the database was built, and the sequence was sequenced; the sequencing data was analyzed through the above-mentioned biological information analysis process and through the method of pattern recognition. Sequencing data shows that the gene methylation levels in the table above have returned to normal levels.
实施例5Example 5
一例肝癌样本，采用本申请的Panel检测，按实施例1的方法采集外周 血；建库，并通过Illumina平台测序；测序数据经上述生物信息的分析流程，得到甲基化水平，结果如下表22所示(表22显示了检测到的大于等于甲基化阈值的靶区域)。A liver cancer sample was detected by the Panel of this application, and peripheral blood was collected according to the method of Example 1; the database was constructed and sequenced on the Illumina platform; the sequencing data was subjected to the above-mentioned biological information analysis process to obtain the methylation level. The results are shown in Table 22 As shown (Table 22 shows the detected target regions greater than or equal to the methylation threshold).
表22Table 22
基因Gene CHRCHR 起始Start 终止termination 甲基化比率Methylation ratio 靶区域序列号Target area serial number
TBX15TBX15 11 119527108119527108 119527157119527157 0.550.55 Seq ID No.63Seq ID No. 63
CRYGDCRYGD 22 208989200208989200 208989249208989249 0.600.60 Seq ID No.64Seq ID No. 64
MTHFD2MTHFD2 22 7442552374425523 7442557274425572 0.60.6 Seq ID No.73Seq ID No. 73
GLI2GLI2 22 121570226121570226 121570275121570275 0.430.43 Seq ID No.74Seq ID No. 74
RASSF1RASSF1 33 5037835950378359 5037843250378432 0.540.54 Seq ID No.15Seq ID No. 15
APCAPC 55 112073300112073300 112073439112073439 0.370.37 Seq ID No.23Seq ID No. 23
TRIM15TRIM15 66 3013100130131001 3013105030131050 0.310.31 Seq ID No.84Seq ID No. 84
TRIM15TRIM15 66 3013170130131701 3013176830131768 0.480.48 Seq ID No.31Seq ID No. 31
HOXA11ASHOXA11AS 77 2722542827225428 2722549727225497 0.40.4 Seq ID No.33Seq ID No. 33
HOXA11ASHOXA11AS 77 2722552327225523 2722557727225577 0.490.49 Seq ID No.34Seq ID No. 34
CCND2CCND2 1212 43817404381740 43818014381801 0.430.43 Seq ID No.51Seq ID No. 51
CCND2CCND2 1212 43818344381834 43818834381883 0.400.40 Seq ID No.52Seq ID No. 52
对检测样本进行模式识别分类鉴定，首先判读出泛癌特异性标志物TBX15和CRYGD基因的甲基化水平大于等于55％和60％，则初步判断该样本为患有癌症的样本；其次，判读出癌症特异性标志物RASSF1、APC、TRIM15、HOXA11AS、CCND2的甲基化水平均大于等于表1中所示的各自阈值(如上表22所示)，则进一步判断该样本为患有以下11种癌症(食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌)中的任意癌症的样本；最后，判读组织特异性标志物，基于表22中大于等于各自阈值的靶区域，可以看出属于肝组织特异性的2个靶区域Seq ID No.73和Seq ID No.74大于等于各自的甲基化水平的阈值，没有其他的组织特异性标志物的甲基化大于等于其各自的阈值，因此在大于等于各自甲基化阈值的组织特异性标志物中，肝组织特异性标志物最多，则最终判断该样本为患有肝癌的样本。Perform pattern recognition classification and identification of the test sample. First, determine the methylation level of pan-cancer specific markers TBX15 and CRYGD genes greater than or equal to 55% and 60%, and then preliminarily determine that the sample is a sample with cancer; secondly, determine and read The methylation levels of the cancer-specific markers RASSF1, APC, TRIM15, HOXA11AS, and CCND2 are greater than or equal to the respective thresholds shown in Table 1 (as shown in Table 22 above), and the sample is further judged to have the following 11 cancers ( Esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and endometrial cancer) samples of any cancer; finally, the interpretation of tissue-specific markers, based on In Table 22, the target regions that are greater than or equal to their respective thresholds, it can be seen that the two target regions Seq ID No. 73 and Seq ID No. 74 that are specific to liver tissue are greater than or equal to their respective thresholds of methylation levels, and there are no other tissues The methylation of specific markers is greater than or equal to their respective thresholds. Therefore, among the tissue-specific markers greater than or equal to their respective methylation thresholds, liver tissue-specific markers are the most, and the sample is finally judged to be a sample with liver cancer .
患者术后48小时，再次抽血，采用本申请的Panel检测，按实施例1的方法采集外周血，建库，测序；测序数据经上述生物信息的分析流程，通过模式识别的方法，分析所有测序数据，结果显示上表中的基因甲基化水平回归正常水平。After 48 hours after the operation of the patient, the blood was drawn again, the Panel test of this application was used, the peripheral blood was collected according to the method of Example 1, the database was built, and the sequence was sequenced; the sequencing data was analyzed through the above-mentioned biological information analysis process and through the method of pattern recognition. Sequencing data shows that the gene methylation levels in the table above have returned to normal levels.
实施例6Example 6
一例食管癌样本，采用本申请的Panel检测，按实施例1的方法采集外周血；建库，并通过Illumina平台测序；测序数据经上述生物信息的分析流程，得到甲基化水平，结果如下表23所示(表23显示了检测到的大于等于甲基化阈值的靶区域)。A sample of esophageal cancer was detected by the Panel of this application, and peripheral blood was collected according to the method of Example 1; the database was built and sequenced on the Illumina platform; the sequencing data was subjected to the above-mentioned biological information analysis process to obtain the methylation level. The results are as follows 23 (Table 23 shows the detected target regions greater than or equal to the methylation threshold).
表23Table 23
Figure PCTCN2021081276-appb-000028
Figure PCTCN2021081276-appb-000028
对检测样本进行模式识别分类鉴定，首先判读出泛癌特异性标志物TBX15和CRYGD基因的甲基化水平大于等于55％和60％，则初步判断该样本为患有癌症的样本；其次，判读出癌症特异性标志物CPE、TFAP2E、TRH、C11orf21、EDNRB的甲基化水平均大于等于表1中所示的各自阈值(如上表23所示)，则进一步判断该样本为患有以下11种癌症(食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌)中的任意癌症的样本；最后，判读组织特异性标志物，基于表23中大于等于各自阈值的靶区域，可以看出属于食管组织特异性的4个靶区域Seq ID No.66、Seq ID No.67、Seq ID No.79和Seq ID No.80大于等于各自的甲基化水平的阈值，没有其他的组织特异性标志物的甲基化大于等于其各自的阈值，因此在大于等于各自甲基化阈值的组织特异性标志物中，食管组织特异性标志物最多，则最终判断该样本为患有食管癌的样本。Perform pattern recognition classification and identification of the test sample. First, determine the methylation level of pan-cancer specific markers TBX15 and CRYGD genes greater than or equal to 55% and 60%, and then preliminarily determine that the sample is a sample with cancer; secondly, determine and read The methylation levels of the cancer-specific markers CPE, TFAP2E, TRH, C11orf21, and EDNRB are greater than or equal to the respective thresholds shown in Table 1 (as shown in Table 23 above), and the sample is further judged to have the following 11 cancers ( Esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and endometrial cancer) samples of any cancer; finally, the interpretation of tissue-specific markers, based on In Table 23, the target regions that are greater than or equal to their respective thresholds, it can be seen that the four target regions that are specific to esophageal tissue, Seq ID No. 66, Seq ID No. 67, Seq ID No. 79, and Seq ID No. 80 are greater than or equal to each The threshold of methylation level, no other tissue-specific markers have methylation greater than or equal to their respective thresholds. Therefore, among the tissue-specific markers greater than or equal to their respective methylation thresholds, esophageal tissue-specific markers At most, the sample is finally judged to be a sample suffering from esophageal cancer.
患者术后48小时，再次抽血，采用本申请的Panel检测，按实施例1的方法采集外周血，建库，测序；测序数据经上述生物信息的分析流程，通过模式识别的方法，分析所有测序数据，结果显示上表中的基因甲基化水平回归正常水平。After 48 hours after the operation of the patient, the blood was drawn again, the Panel test of this application was used, the peripheral blood was collected according to the method of Example 1, the database was built, and the sequence was sequenced; the sequencing data was analyzed through the above-mentioned biological information analysis process and through the method of pattern recognition. Sequencing data shows that the gene methylation levels in the table above have returned to normal levels.
实施例7Example 7
一例胰腺癌样本，采用本申请的Panel检测，按实施例1的方法采集外周血；建库，并通过Illumina平台测序；测序数据经上述生物信息的分析流程，得到甲基化水平，结果如下表24所示(表24显示了检测到的大于等于甲基化阈值的靶区域)。A sample of pancreatic cancer was detected by the Panel of this application, and peripheral blood was collected according to the method in Example 1; the database was constructed and sequenced on the Illumina platform; the sequencing data was subjected to the above-mentioned biological information analysis process to obtain the methylation level. The results are as follows 24 (Table 24 shows the detected target regions greater than or equal to the methylation threshold).
表24Table 24
基因Gene CHRCHR 起始Start 终止termination 甲基化比率Methylation ratio 靶区域序列号Target area serial number
TBX15TBX15 11 119527108119527108 119527157119527157 0.550.55 Seq ID No.63Seq ID No. 63
CRYGDCRYGD 22 208989200208989200 208989249208989249 0.600.60 Seq ID No.64Seq ID No. 64
FRYFRY 1313 3260535832605358 3260540732605407 0.320.32 Seq ID No.111Seq ID No.111
FRYFRY 1313 3260556332605563 3260561232605612 0.400.40 Seq ID No.112Seq ID No. 112
FRYFRY 1313 3260590232605902 3260595132605951 0.390.39 Seq ID No.113Seq ID No. 113
BRF1BRF1 1414 105714785105714785 105714844105714844 0.330.33 Seq ID No.114Seq ID No. 114
BRF1BRF1 1414 105715025105715025 105715074105715074 0.380.38 Seq ID No.115Seq ID No. 115
HOPXHOPX 44 5752244557522445 5752249457522494 0.600.60 Seq ID No.18Seq ID No. 18
SFRP2SFRP2 44 154710475154710475 154710536154710536 0.380.38 Seq ID No.19Seq ID No.19
SFRP2SFRP2 44 154710598154710598 154710647154710647 0.390.39 Seq ID No.20Seq ID No. 20
SFRP2SFRP2 44 154710702154710702 154710751154710751 0.470.47 Seq ID No.21Seq ID No. 21
SFRP2SFRP2 44 154710796154710796 154710845154710845 0.530.53 Seq ID No.22Seq ID No. 22
GFRA1GFRA1 1010 118032831118032831 118032906118032906 0.690.69 Seq ID No.40Seq ID No. 40
GFRA1GFRA1 1010 118032948118032948 118032997118032997 0.370.37 Seq ID No.41Seq ID No. 41
HOXB4HOXB4 1717 4665533946655339 4665539546655395 0.420.42 Seq ID No.59Seq ID No. 59
HOXB4HOXB4 1717 4665542846655428 4665547746655477 0.610.61 Seq ID No.60Seq ID No. 60
SALL1SALL1 1616 5118437951184379 5118444151184441 0.630.63 Seq ID No.58Seq ID No. 58
对检测样本进行模式识别分类鉴定，首先判读出泛癌特异性标志物TBX15和CRYGD基因的甲基化水平大于等于55％和60％，则初步判断该样本为患有癌症的样本；其次，判读出癌症特异性标志物HOPX、SFRP2、GFRA1、HOXB4、SALL1的甲基化水平均大于等于表1中所示的各自阈值(如上表24所示)，则进一步判断该样本为患有以下11种癌症(食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌)中的任意癌症的样本；最后，判读组织特异性标志物，基 于表24中大于等于各自阈值的靶区域，可以看出属于胰腺组织特异性的5个靶区域Seq ID No.111、Seq ID No.112、Seq ID No.113、Seq ID No.114和Seq ID No.115大于等于各自的甲基化水平的阈值，没有其他的组织特异性标志物的甲基化大于等于其各自的阈值，因此在大于等于各自甲基化阈值的组织特异性标志物中，胰腺组织特异性标志物最多，则最终判断该样本为患有胰腺癌的样本。Perform pattern recognition classification and identification of the test sample. Firstly, determine the methylation levels of pan-cancer specific markers TBX15 and CRYGD genes greater than or equal to 55% and 60%, and then preliminarily determine that the sample is a sample suffering from cancer; The methylation levels of the cancer-specific markers HOPX, SFRP2, GFRA1, HOXB4, and SALL1 are greater than or equal to the respective thresholds shown in Table 1 (as shown in Table 24 above), and the sample is further judged to have the following 11 cancers ( Esophageal cancer, stomach cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and endometrial cancer) samples of any cancer; finally, the interpretation of tissue-specific markers, based on In Table 24, the target regions that are greater than or equal to their respective thresholds can be seen as five target regions Seq ID No. 111, Seq ID No. 112, Seq ID No. 113, Seq ID No. 114, and Seq ID that are specific to pancreatic tissue No. 115 is greater than or equal to the respective threshold of methylation level, and no other tissue-specific markers have methylation greater than or equal to their respective thresholds. Therefore, among tissue-specific markers greater than or equal to their respective methylation thresholds, The pancreatic tissue-specific markers are the most, and the sample is finally judged to be a sample with pancreatic cancer.
患者术后48小时，再次抽血，采用本申请的Panel检测，按实施例1的方法采集外周血，建库，测序；测序数据经上述生物信息的分析流程，通过模式识别的方法，分析所有测序数据，结果显示上表中的基因甲基化水平回归正常水平。After 48 hours after the operation of the patient, the blood was drawn again, the Panel test of this application was used, the peripheral blood was collected according to the method of Example 1, the database was built, and the sequence was sequenced; the sequencing data was analyzed through the above-mentioned biological information analysis process and through the method of pattern recognition. Sequencing data shows that the gene methylation levels in the table above have returned to normal levels.
实施例8Example 8
一例乳腺癌样本，采用本申请的Panel检测，按实施例1的方法采集外周血；建库，并通过Illumina平台测序；测序数据经上述生物信息的分析流程，得到甲基化水平，结果如下表25所示(表25显示了检测到的大于等于甲基化阈值的靶区域)。A breast cancer sample was detected by the Panel of this application, and peripheral blood was collected according to the method of Example 1; the database was constructed and sequenced on the Illumina platform; the sequencing data was subjected to the above-mentioned biological information analysis process to obtain the methylation level. The results are as follows 25 (Table 25 shows the detected target regions greater than or equal to the methylation threshold).
表25Table 25
基因Gene CHRCHR 起始Start 终止termination 甲基化比率Methylation ratio 靶区域序列号Target area serial number
TBX15TBX15 11 119527108119527108 119527157119527157 0.550.55 Seq ID No.63Seq ID No. 63
CRYGDCRYGD 22 208989200208989200 208989249208989249 0.600.60 Seq ID No.64Seq ID No. 64
SLC2A2SLC2A2 33 170746025170746025 170746074170746074 0.540.54 Seq ID No.78Seq ID No. 78
LEMD2LEMD2 66 3373935833739358 3373940733739407 0.490.49 Seq ID No.85Seq ID No. 85
LEMD2LEMD2 66 3373955833739558 3373960733739607 0.490.49 Seq ID No.86Seq ID No. 86
TRIM15TRIM15 66 3013100130131001 3013105030131050 0.620.62 Seq ID No.84Seq ID No. 84
RUNX3RUNX3 11 2525745725257457 2525758825257588 0.790.79 Seq ID No.1Seq ID No. 1
RUNX3RUNX3 11 2525813325258133 2525819525258195 0.660.66 Seq ID No.2Seq ID No. 2
RUNX3RUNX3 11 2525822525258225 2525828525258285 0.710.71 Seq ID No.3Seq ID No. 3
TFAP2ETFAP2E 11 3604293836042938 3604298736042987 0.670.67 Seq ID No.4Seq ID No. 4
TFAP2ETFAP2E 11 3604300236043002 3604306336043063 0.730.73 Seq ID No.5Seq ID No. 5
TFAP2ETFAP2E 11 3604358536043585 3604363436043634 0.660.66 Seq ID No.6Seq ID No. 6
RASSF1RASSF1 33 5037835950378359 5037843250378432 0.540.54 Seq ID No.15Seq ID No. 15
SFRP2SFRP2 44 154710475154710475 154710536154710536 0.380.38 Seq ID No.19Seq ID No.19
SFRP2SFRP2 44 154710598154710598 154710647154710647 0.450.45 Seq ID No.20Seq ID No. 20
SFRP2SFRP2 44 154710702154710702 154710751154710751 0.540.54 Seq ID No.21Seq ID No. 21
SFRP2SFRP2 44 154710796154710796 154710845154710845 0.430.43 Seq ID No.22Seq ID No. 22
CCND2CCND2 1212 43817404381740 43818014381801 0.470.47 Seq ID No.51Seq ID No. 51
CCND2CCND2 1212 43818344381834 43818834381883 0.550.55 Seq ID No.52Seq ID No. 52
对检测样本进行模式识别分类鉴定，首先判读出泛癌特异性标志物TBX15和CRYGD基因的甲基化水平大于等于55％和60％，则初步判断该样本为患有癌症的样本；其次，判读出癌症特异性标志物TRIM15、RUNX3、TFAP2E、RASSF1、SFRP2、CCND2的甲基化水平均大于等于表1中所示的各自阈值(如上表25所示)，则进一步判断该样本为患有以下11种癌症(食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌)中的任意癌症的样本；最后，判读组织特异性标志物，基于表25中大于等于各自阈值的靶区域，可以看出属于乳腺组织特异性的3个靶区域Seq ID No.78、Seq ID No.85和Seq ID No.86大于等于各自的甲基化水平的阈值，没有其他的组织特异性标志物的甲基化大于等于其各自的阈值，因此在大于等于各自甲基化阈值的组织特异性标志物中，乳腺组织特异性标志物最多，则最终判断该样本为患有乳腺癌的样本。Perform pattern recognition classification and identification of the test sample. First, determine the methylation level of pan-cancer specific markers TBX15 and CRYGD genes greater than or equal to 55% and 60%, and then preliminarily determine that the sample is a sample with cancer; secondly, determine and read The methylation levels of cancer-specific markers TRIM15, RUNX3, TFAP2E, RASSF1, SFRP2, and CCND2 are greater than or equal to the respective thresholds shown in Table 1 (as shown in Table 25 above), and then the sample is further judged to have the following 11 types A sample of any cancer (esophageal cancer, stomach cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer); finally, interpret tissue-specific markers Based on the target regions that are greater than or equal to their respective thresholds in Table 25, it can be seen that the three target regions that are specific to breast tissue, Seq ID No. 78, Seq ID No. 85, and Seq ID No. 86, are greater than or equal to their respective methylation. No other tissue-specific markers have methylation greater than or equal to their respective thresholds. Therefore, among the tissue-specific markers greater than or equal to their respective methylation thresholds, breast tissue-specific markers are the most, and the final The sample is judged to be a sample suffering from breast cancer.
患者术后48小时，再次抽血，采用本申请的Panel检测，按实施例1的方法采集外周血，建库，测序；测序数据经上述生物信息的分析流程，通过模式识别的方法，分析所有测序数据，结果显示上表中的基因甲基化水平回归正常水平。After 48 hours after the operation of the patient, the blood was drawn again, the Panel test of this application was used, the peripheral blood was collected according to the method of Example 1, the database was built, and the sequence was sequenced; the sequencing data was analyzed through the above-mentioned biological information analysis process and through the method of pattern recognition. Sequencing data shows that the gene methylation levels in the table above have returned to normal levels.
对于本领域的技术人员来说，可以根据以上的技术方案和构思，作出各种相应的改变和变形，而所有的这些改变和变形都应该包括在本申请权利要求的保护范围之内。For those skilled in the art, various corresponding changes and modifications can be made based on the above technical solutions and concepts, and all these changes and modifications should be included in the protection scope of the claims of this application.

Claims (21)

  1. 一种探针组合物，其包括：A probe composition comprising:
    靶向食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌中的任意癌症的特异性区域的探针，Probes targeting specific regions of any cancer among esophageal cancer, stomach cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, and endometrial cancer,
    其中，所述癌症特异性区域选自Seq ID No.：1-62中的任意。Wherein, the cancer-specific region is selected from any of Seq ID No.: 1-62.
  2. 根据权利要求1所述的探针组合物，其还包括：The probe composition of claim 1, further comprising:
    靶向泛癌特异性区域中的任意的探针，Target any probe in the pan-cancer specific region,
    所述泛癌特异性区域选自Seq ID No.：63-64中的任意。The pan-cancer specific region is selected from any of Seq ID No.: 63-64.
  3. 根据权利要求1所述的探针组合物，其还包括：The probe composition of claim 1, further comprising:
    靶向组织特异性区域中的任意的探针，Target any probe in the specific area of the tissue,
    所述组织特异性区域选自Seq ID No.：65-117中的任意。The tissue-specific region is selected from any of Seq ID No.: 65-117.
  4. 根据权利要求3所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：66-67、79、81-82、97-102、116-117是食管的组织特异性靶区域。The probe composition according to claim 3, wherein Seq ID No.: 66-67, 79, 81-82, 97-102, 116-117 in the tissue-specific region are tissue-specific of the esophagus Sexual target area.
  5. 根据权利要求3所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：81、82、97-102是胃的组织特异性靶区域。The probe composition according to claim 3, wherein Seq ID Nos.: 81, 82, 97-102 in the tissue-specific region are tissue-specific target regions of the stomach.
  6. 根据权利要求3所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：83、87-90、110是结直肠的组织特异性靶区域。The probe composition according to claim 3, wherein Seq ID No. 83, 87-90, 110 in the tissue-specific region are tissue-specific target regions of the colorectal.
  7. 根据权利要求3所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：65、70-72、76、91-92、103-104是肺的组织特异性靶区域。The probe composition of claim 3, wherein Seq ID No. 65, 70-72, 76, 91-92, 103-104 in the tissue-specific region are tissue-specific targets of the lung area.
  8. 根据权利要求3所述的探针组合物，其特征在于，所述组织特异性区域中：Seq ID No.：73-74、107是肝的组织特异性靶区域。The probe composition according to claim 3, wherein in the tissue-specific region: Seq ID No.: 73-74 and 107 are tissue-specific target regions of the liver.
  9. 根据权利要求3所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：111-115是胰腺的组织特异性靶区域。The probe composition of claim 3, wherein Seq ID No.: 111-115 in the tissue-specific region is a tissue-specific target region of the pancreas.
  10. 根据权利要求3所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：80、84、93、95是前列腺的组织特异性靶区域。The probe composition according to claim 3, wherein Seq ID No. 80, 84, 93, 95 in the tissue-specific region are tissue-specific target regions of the prostate.
  11. 根据权利要求3所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：78、84-86是乳腺的组织特异性靶区域。The probe composition according to claim 3, wherein Seq ID Nos.: 78 and 84-86 in the tissue-specific region are tissue-specific target regions of the breast.
  12. 根据权利要求3所述的探针组合物，其特征在于，在所述组织特异 性区域中，Seq ID No.：77、96、105是卵巢的组织特异性靶区域。The probe composition according to claim 3, wherein in the tissue-specific region, Seq ID No.: 77, 96, and 105 are tissue-specific target regions of the ovary.
  13. 根据权利要求3所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：68-69、75、109是宫颈的组织特异性靶区域。The probe composition according to claim 3, wherein Seq ID No.: 68-69, 75, and 109 in the tissue-specific region are tissue-specific target regions of the cervix.
  14. 根据权利要求3所述的探针组合物，其特征在于，所述组织特异性区域中Seq ID No.：94、106、108是子宫内膜的组织特异性靶区域。The probe composition according to claim 3, wherein Seq ID No. 94, 106, 108 in the tissue-specific region are tissue-specific target regions of the endometrium.
  15. 根据权利要求1-14中任一项所述的探针组合物，其包括：The probe composition according to any one of claims 1-14, which comprises:
    低甲基化探针，其与经重亚硫酸盐转化的不含CG甲基化的所述特异性区域杂交，和A hypomethylation probe that hybridizes to the specific region that is converted by bisulfite and does not contain CG methylation, and
    高甲基化探针，其与经重亚硫酸盐转化的CG全部甲基化的所述特异性区域杂交。A hypermethylated probe that hybridizes to the specific region where the bisulfite-converted CG is fully methylated.
  16. 根据权利要求15所述的探针组合物，其中每一个探针的长度为40～60bp。The probe composition according to claim 15, wherein the length of each probe is 40-60 bp.
  17. 根据权利要求16所述的探针组合物，其中每一个探针的长度为45～56bp，优选50～56bp，进一步优选50bp。The probe composition according to claim 16, wherein the length of each probe is 45-56 bp, preferably 50-56 bp, and more preferably 50 bp.
  18. 根据权利要求15所述的探针组合物，其特征在于，所述低甲基化探针包括靶向癌症特异性区域的探针Seq ID No.：118-214中的任意，靶向泛癌特异性区域的探针Seq ID No.：215-216中的任意，和靶向组织特异性区域的探针Seq ID No.：217-274中的任意。The probe composition according to claim 15, wherein the hypomethylation probe comprises a probe targeting a cancer-specific region. Seq ID No.: any of 118-214, targeting pan-cancer Seq ID No. of probes for specific regions: any of 215-216, and Seq ID No. of probes for tissue-specific regions: any of 217-274.
  19. 根据权利要求15所述的探针组合物，其特征在于，所述高甲基化探针包括靶向癌症特异性区域的探针Seq ID No.：275-371中的任意，靶向泛癌特异性区域的探针Seq ID No.：372-373中的任意，和靶向组织特异性区域的探针Seq ID No.：374-431中的任意。The probe composition according to claim 15, wherein the hypermethylation probe comprises a probe targeting a cancer-specific region Seq ID No.: any of 275-371, targeting pan-cancer specificity The probe Seq ID No. of the region: any of 372-373, and the probe Seq ID No. of the tissue-specific region: any of 374-431.
  20. 一种试剂盒，其包括权利要求1-19中任一项所述的探针组合物。A kit comprising the probe composition according to any one of claims 1-19.
  21. 一种权利要求1-19中任一项所述的探针组合物在制备检测食管癌、胃癌、结直肠癌、肺癌、肝癌、胰腺癌、前列腺癌、乳腺癌、卵巢癌、宫颈癌和子宫内膜癌的试剂盒中的应用。A probe composition according to any one of claims 1-19 is prepared to detect esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer and uterine cancer. Application of the kit for endometrial cancer.
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