WO2021185274A1 - Probe composition for detecting 6 cancers with high incidence in china - Google Patents

Probe composition for detecting 6 cancers with high incidence in china Download PDF

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WO2021185274A1
WO2021185274A1 PCT/CN2021/081275 CN2021081275W WO2021185274A1 WO 2021185274 A1 WO2021185274 A1 WO 2021185274A1 CN 2021081275 W CN2021081275 W CN 2021081275W WO 2021185274 A1 WO2021185274 A1 WO 2021185274A1
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seq
cancer
probe
tissue
specific
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PCT/CN2021/081275
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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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    • C—CHEMISTRY; METALLURGY
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    • 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
    • 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 article relates to a cancer gene methylation detection composition, in particular to a probe composition that specifically recognizes the DNA sequence after Bisulfite treatment, and the detection of esophagus based on the 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 variation 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 is free and extracellular in circulating blood. 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 article provides a probe composition that can be used for the early stage of six cancers including esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer and pancreatic cancer. Screening.
  • 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.
  • this article involves the following:
  • a probe composition comprising: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, wherein the cancer-specific region is selected From Seq ID No.: Any of 1-62.
  • the probe composition according to item 1 further comprising: a probe targeting a pan-cancer specific region, the pan-cancer specific region selected from any of Seq ID No.: 63-64.
  • the probe composition according to item 3 characterized in that Seq ID No.: 66-67, 74-76, 84-89, 99-100 in the tissue-specific region are tissue-specific of the esophagus Sexual target area.
  • the probe composition according to item 3 characterized in that, in the tissue-specific region, Seq ID No.: 75, 76, 84-89 are tissue-specific target regions of the stomach.
  • the probe composition according to item 3 characterized in that, in the tissue-specific region, Seq ID No.: 77-81, 93 are tissue-specific target regions of the colorectal.
  • the probe composition according to item 3 characterized in that, in the tissue-specific region, Seq ID No.: 65, 68-70, 73, 82-83, 90-91 are pulmonary Tissue specific target area.
  • the probe composition according to item 3 characterized in that, in the tissue-specific region, Seq ID No.: 71-72, 92 are tissue-specific target regions of the liver.
  • the probe composition according to any one of items 1-9 which comprises: a hypomethylation probe, which is specific to the bisulfite-converted cancer without CG methylation Hybridization of sexual regions, pan-cancer-specific regions, and tissue-specific regions, and; hypermethylation probes, which are all methylated with bisulfite-converted CG, the cancer-specific regions and pan-cancer-specific regions , And tissue-specific region hybridization.
  • each probe in the probe composition is 40-60 bp in length.
  • Seq ID No. any of 101-197
  • Seq ID No. of probes for tissue-specific regions any of 200-240.
  • a kit comprising the probe composition according to any one of items 1-14.
  • This article also provides a method for detecting six cancers, including esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, by using the probe composition.
  • This article also provides a method for simultaneously detecting changes in the methylation level of the above 6 cancers by using the kit.
  • This application also provides a method for simultaneously detecting changes in the methylation level of the above six cancers 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.
  • one Panel can complete the screening of major cancers, which saves the cost of probe synthesis, simplifies the experimental process, and facilitates the operation of experimenters.
  • FIG 1 shows the implementation process of this article
  • This article 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: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, wherein, The cancer-specific region is selected from any of Seq ID No.: 1-62; a probe targeting a pan-cancer specific region, and the pan-cancer specific region is selected from Seq ID No.: 63-64 Any; and a probe targeting a tissue-specific region, the tissue-specific region is selected from any of Seq ID No.: 65-100.
  • Seq ID No.: 66-67, 74-76, 84-89, and 99-100 are tissue-specific target regions of the esophagus.
  • a probe composition includes: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, wherein, The cancer-specific region is selected from any of Seq ID No.: 1-62; a probe targeting a pan-cancer specific region, and the pan-cancer specific region is selected from Seq ID No.: 63-64 Any; and a probe targeting a tissue-specific region, the tissue-specific region is selected from any of Seq ID No.: 65-100.
  • Seq ID No.: 75, 76, 84-89 are tissue-specific target regions of the stomach.
  • a probe composition includes: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, wherein, The cancer-specific region is selected from any of Seq ID No.: 1-62; a probe targeting a pan-cancer specific region, and the pan-cancer specific region is selected from Seq ID No.: 63-64 Any; and a probe targeting a tissue-specific region, the tissue-specific region is selected from any of Seq ID No.: 65-100.
  • Seq ID No.: 77-81 and 93 are tissue-specific target regions of the colorectal.
  • a probe composition includes: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, wherein, The cancer-specific region is selected from any of Seq ID No.: 1-62; a probe targeting a pan-cancer specific region, and the pan-cancer specific region is selected from Seq ID No.: 63-64 Any; and a probe targeting a tissue-specific region, the tissue-specific region is selected from any of Seq ID No.: 65-100.
  • Seq ID No.: 65, 68-70, 73, 82-83, and 90-91 are tissue-specific target regions of the lung.
  • a probe composition includes: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, wherein, The cancer-specific region is selected from any of Seq ID No.: 1-62; a probe targeting a pan-cancer specific region, and the pan-cancer specific region is selected from Seq ID No.: 63-64 Any; and a probe targeting a tissue-specific region, the tissue-specific region is selected from any of Seq ID No.: 65-100.
  • Seq ID No.: 71-72, 92 are tissue-specific target regions of the liver.
  • a probe composition includes: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, wherein, The cancer-specific region is selected from any of Seq ID No.: 1-62; a probe targeting a pan-cancer specific region, and the pan-cancer specific region is selected from Seq ID No.: 63-64 Any; and a probe targeting a tissue-specific region, the tissue-specific region is selected from any of Seq ID No.: 65-100.
  • Seq ID No.: 94-98 is the tissue-specific target region of the pancreas.
  • the above-mentioned probe composition includes a hypomethylation probe, which is different from the cancer-specific region and pan-cancer-specific region converted by bisulfite without CG methylation.
  • the hypomethylation probes include probes targeting cancer-specific regions Seq ID No.: any of 101-197, and probes targeting pan-cancer specific regions Seq ID No.: 198-199 Any, and Seq ID No. of probes targeting tissue-specific regions: Any of 200-240.
  • the hypermethylation probes include probes targeting cancer-specific regions, Seq ID No.: 241-337, and probes targeting pan-cancer specific regions, Seq ID No.: 338-339, Seq ID No.: any of 340-380 probes targeting tissue-specific regions.
  • Seq ID No. 101, Seq ID No. 102, and Seq ID No. 103 are all hypomethylated probes that target the target region shown in Seq ID No. 1
  • Seq ID No. 241 , Seq ID No. 242 and Seq ID No. 243 are hypermethylated probes that target the target region shown in Seq ID No. 1.
  • Seq ID No. 104 and Seq ID No. 105 are hypomethylated probes that target the target region shown in Seq ID No. 2, and Seq ID No. 244 and Seq ID No. 245 are both targeted to Seq ID No. .2 Hypermethylated probes in the target region shown.
  • Seq ID No. 107 are hypomethylated probes that target the target region shown in Seq ID No. 3, and Seq ID No. 246 and Seq ID No. 247 are both targeted to Seq ID No. .3 Hypermethylated probes in the target region shown.
  • Seq ID No. 108 is a hypomethylated probe targeting the target region shown in Seq ID No. 4
  • Seq ID No. 248 is a hypermethylated probe targeting the target region shown in Seq ID No. 4.
  • Seq ID No. 109 and Seq ID No. 110 are hypomethylated probes that target the target region shown in Seq ID No. 5, and Seq ID No. 249 and Seq ID No. 250 are both targeted to Seq ID No. .3 Hypermethylated probes in the target region shown.
  • Seq ID No. 112 is a hypomethylated probe targeting the target region shown in Seq ID No. 7
  • Seq ID No. 252 is a hypermethylated probe targeting the target region shown in Seq ID No. 7.
  • Seq ID No. 113 and Seq ID No. 114 are hypomethylated probes that target the target region shown in Seq ID No. 8, and Seq ID No. 253 and Seq ID No. 254 are both targeted to Seq ID No.
  • Seq ID No. 116, and Seq ID No. 117 are all hypomethylated probes that target the target region shown in Seq ID No. 9, Seq ID No. 255, Seq ID No. 256 and Seq ID No. 256.
  • Seq ID No. 257 is a hypermethylated probe that targets the target region shown in Seq ID No. 9.
  • Seq ID No.118 is a hypomethylated probe targeting the target region shown in Seq ID No.10
  • Seq ID No.258 is a hypermethylated probe targeting the target region shown in Seq ID No.10.
  • Seq ID No. 119 is a hypomethylated probe targeting the target region shown in Seq ID No. 11, and Seq ID No. 259 is a hypermethylated probe targeting the target region shown in Seq ID No. 11.
  • Seq ID No. 120 is a hypomethylated probe targeting the target region shown in Seq ID No. 12, and Seq ID No. 260 is a hypermethylated probe targeting the target region shown in Seq ID No. 12.
  • Seq ID No. 121 is a hypomethylated probe targeting the target region shown in Seq ID No. 13, and Seq ID No. 261 is a hypermethylated probe targeting the target region shown in Seq ID No. 13.
  • Seq ID No. 122 and Seq ID No. 123 are hypomethylation probes that target the target region shown in Seq ID No. 14, and Seq ID No. 262 and Seq ID No. 263 are both targeted to Seq ID No.
  • Seq ID No. 125 are hypomethylated probes that target the target region shown in Seq ID No. 15, and Seq ID No. 264 and Seq ID No. 265 are both targeted to Seq ID No.
  • Seq ID No. 126 and Seq ID No. 127 are both hypomethylated probes that target the target region shown in Seq ID No. 16, and Seq ID No. 266 and Seq ID No. 267 are both targeted to Seq ID No.
  • Seq ID No. 128 and Seq ID No. 129 are both hypomethylated probes that target the target region shown in Seq ID No. 17, and Seq ID No. 268 and Seq ID No.
  • Seq ID No. 130 is a hypomethylated probe targeting the target region shown in Seq ID No. 18, and Seq ID No. 270 is a hypermethylated probe targeting the target region shown in Seq ID No. 18.
  • Seq ID No. 131 and Seq ID No. 132 are hypomethylated probes that target the target region shown in Seq ID No. 19, and Seq ID No. 271 and Seq ID No. 272 are both targeted to Seq ID No.
  • Seq ID No. 133 is a hypomethylated probe targeting the target region shown in Seq ID No. 20, and Seq ID No.
  • Seq ID No. 273 is a hypermethylated probe targeting the target region shown in Seq ID No. 20.
  • Seq ID No. 134 is a hypomethylated probe targeting the target region shown in Seq ID No. 21, and Seq ID No. 274 is a hypermethylated probe targeting the target region shown in Seq ID No. 21.
  • Seq ID No. 135 is a hypomethylated probe targeting the target region shown in Seq ID No. 22, and Seq ID No. 275 is a hypermethylated probe targeting the target region shown in Seq ID No. 22.
  • Seq ID No. 136, Seq ID No. 137, and Seq ID No. 138 are hypomethylated probes that target the target region shown in Seq ID No. 23, Seq ID No. 276, Seq ID No.
  • Seq ID No. 278 are all hypermethylated probes that target the target region shown in Seq ID No. 23.
  • Seq ID No. 139 and Seq ID No. 140 are both hypomethylated probes that target the target region shown in Seq ID No. 24, and Seq ID No. 279 and Seq ID No. 280 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. 25, and Seq ID No. 281 and Seq ID No. 282 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. 26, and Seq ID No. 283 and Seq ID No. 284 are both targeted to Seq ID No.
  • Seq ID No. 145 is a hypomethylated probe targeting the target region shown in Seq ID No. 27, and Seq ID No. 285 is a hypermethylated probe targeting the target region shown in Seq ID No. 27.
  • Seq ID No. 146 and Seq ID No. 147 are hypomethylated probes that target the target region shown in Seq ID No. 28, and Seq ID No. 286 and Seq ID No. 287 are both targeted to Seq ID No.
  • the hypermethylated probe in the target region shown in .28. Seq ID No. 148 and Seq ID No. 149 are both hypomethylated probes that target the target region shown in Seq ID No. 29, and Seq ID No. 288 and Seq ID No. 289 are both targeted to Seq ID No.
  • Seq ID No. 150 is a hypomethylated probe that targets the target region shown in Seq ID No. 30, and Seq ID No. 290 is a hypermethylated probe that targets the target region shown in Seq ID No. 30.
  • Seq ID No. 151 and Seq ID No. 152 are both hypomethylated probes that target the target region shown in Seq ID No. 31, and Seq ID No.
  • Seq ID No. 291 and Seq ID No. 292 are both targeted to Seq ID No.
  • Seq ID No. 153 is a hypomethylated probe targeting the target region shown in Seq ID No. 32
  • Seq ID No. 293 is a hypermethylated probe targeting the target region shown in Seq ID No. 32.
  • Seq ID No. 154 and Seq ID No. 155 are hypomethylated probes that target the target region shown in Seq ID No. 33
  • Seq ID No. 294 and Seq ID No. 295 are both targeted to Seq ID No.
  • Seq ID No.156 is a hypomethylated probe targeting the target region shown in Seq ID No.34
  • Seq ID No.296 is a hypermethylated probe targeting the target region shown in Seq ID No.34.
  • Seq ID No. 157 and Seq ID No. 158 are hypomethylation probes that target the target region shown in Seq ID No. 35, and Seq ID No. 297 and Seq ID No. 298 are both targeted to Seq ID No.
  • Seq ID No. 159 is a hypomethylated probe targeting the target region shown in Seq ID No. 36
  • Seq ID No. 299 is a hypermethylated probe targeting the target region shown in Seq ID No. 36.
  • Seq ID No. 161 are hypomethylated probes that target the target region shown in Seq ID No. 37, and Seq ID No. 300 and Seq ID No. 301 are both targeted to Seq ID No.
  • Seq ID No. 162 and Seq ID No. 163 are hypomethylation probes that target the target region shown in Seq ID No. 38, and Seq ID No. 302 and Seq ID No. 303 are both targeted to Seq ID No.
  • Seq ID No.164 is a hypomethylated probe targeting the target region shown in Seq ID No.39
  • Seq ID No.304 is a hypermethylated probe targeting the target region shown in Seq ID No.39
  • Seq ID No. 165 and Seq ID No. 166 are hypomethylated probes that target the target region shown in Seq ID No. 40
  • Seq ID No. 305 and Seq ID No. 306 are both targeted to Seq ID No.
  • Seq ID No. 167 is a hypomethylated probe targeting the target region shown in Seq ID No. 41
  • Seq ID No. 307 is a hypermethylated probe targeting the target region shown in Seq ID No. 41.
  • Seq ID No.168 is a hypomethylated probe targeting the target region shown in Seq ID No.42
  • Seq ID No.308 is a hypermethylated probe targeting the target region shown in Seq ID No.42.
  • Seq ID No. 169 is a hypomethylated probe targeting the target region shown in Seq ID No. 43
  • Seq ID No. 309 is a hypermethylated probe targeting the target region shown in Seq ID No. 43.
  • Seq ID No. 170 is a hypomethylated probe targeting the target region shown in Seq ID No. 44
  • Seq ID No. 310 is a hypermethylated probe targeting the target region shown in Seq ID No. 44.
  • Seq ID No. 173 is a hypomethylated probe targeting the target region shown in Seq ID No. 46
  • Seq ID No. 313 is a hypermethylated probe targeting the target region shown in Seq ID No. 46.
  • Seq ID No. 174 is a hypomethylated probe that targets the target region shown in Seq ID No. 47
  • Seq ID No. 314 is a hypermethylated probe that targets the target region shown in Seq ID No. 47.
  • Seq ID No. 175 is a hypomethylated probe targeting the target region shown in Seq ID No. 48
  • Seq ID No. 315 is a hypermethylated probe targeting the target region shown in Seq ID No. 48
  • Seq ID No. 176 is a hypomethylated probe that targets the target region shown in Seq ID No. 49
  • Seq ID No. 316 is a hypermethylated probe that targets the target region shown in Seq ID No. 49
  • Seq ID No. 177 is a hypomethylated probe targeting the target region shown in Seq ID No. 50
  • Seq ID No. 317 is a hypermethylated probe targeting the target region shown in Seq ID No. 50.
  • Seq ID No. 180 is a hypomethylated probe targeting the target region shown in Seq ID No. 52
  • Seq ID No. 320 is a hypermethylated probe targeting the target region shown in Seq ID No. 52.
  • Seq ID No. 181 and Seq ID No. 182 are hypomethylation probes that target the target region shown in Seq ID No. 53, and Seq ID No. 321 and Seq ID No. 322 are both targeted to Seq ID No.
  • Seq ID No. 183 is a hypomethylated probe targeting the target region shown in Seq ID No. 54
  • Seq ID No. 323 is a hypermethylated probe targeting the target region shown in Seq ID No. 54.
  • Seq ID No. 184 and Seq ID No. 185 are hypomethylated probes that target the target region shown in Seq ID No. 55
  • Seq ID No. 324 and Seq ID No. 325 are both targeted to Seq ID No.
  • Seq ID No. 186 and Seq ID No. 187 are hypomethylated probes that target the target region shown in Seq ID No. 56
  • Seq ID No. 326 and Seq ID No. 327 are both targeted to Seq ID No.
  • Seq ID No. 188 and Seq ID No. 189 are both hypomethylated probes that target the target region shown in Seq ID No. 57, and Seq ID No. 328 and Seq ID No. 329 are both targeted to Seq ID No.
  • Seq ID No. 190 and Seq ID No. 191 are hypomethylated probes that target the target region shown in Seq ID No. 58, and Seq ID No. 330 and Seq ID No. 331 are both targeted to Seq ID No.
  • Seq ID No. 193 are both hypomethylated probes that target the target region shown in Seq ID No. 59, and Seq ID No. 332 and Seq ID No. 333 are both targeted to Seq ID No.
  • Seq ID No. 194 is a hypomethylated probe targeting the target region shown in Seq ID No. 60
  • Seq ID No. 334 is a hypermethylated probe targeting the target region shown in Seq ID No. 60.
  • Seq ID No. 195 and Seq ID No. 196 are hypomethylated probes that target the target region shown in Seq ID No. 61, and Seq ID No. 335 and Seq ID No. 336 are both targeted to Seq ID No.
  • Seq ID No.197 is a hypomethylated probe targeting the target region shown in Seq ID No.62
  • Seq ID No.337 is a hypermethylated probe targeting the target region shown in Seq ID No.62.
  • Seq ID No. 198 is a hypomethylated probe targeting the target region shown in Seq ID No. 63
  • Seq ID No. 338 is a hypermethylated probe targeting the target region shown in Seq ID No. 63.
  • Seq ID No. 199 is a hypomethylated probe targeting the target region shown in Seq ID No. 64
  • Seq ID No. 339 is a hypermethylated probe targeting the target region shown in Seq ID No. 64.
  • Seq ID No. 200 is a hypomethylated probe targeting the target region shown in Seq ID No. 65
  • Seq ID No. 340 is a hypermethylated probe targeting the target region shown in Seq ID No. 65.
  • Seq ID No. 201 is a hypomethylated probe targeting the target region shown in Seq ID No. 66
  • Seq ID No. 341 is a hypermethylated probe targeting the target region shown in Seq ID No. 66.
  • Seq ID No. 202 is a hypomethylated probe targeting the target region shown in Seq ID No. 67
  • Seq ID No. 342 is a hypermethylated probe targeting the target region shown in Seq ID No. 67.
  • Seq ID No. 203 is a hypomethylated probe targeting the target region shown in Seq ID No.
  • Seq ID No. 206 is a hypomethylated probe targeting the target region shown in Seq ID No. 71, and Seq ID No.
  • Seq ID No. 346 is a hypermethylated probe targeting the target region shown in Seq ID No. 71.
  • Seq ID No. 207 is a hypomethylated probe targeting the target region shown in Seq ID No. 72
  • Seq ID No. 347 is a hypermethylated probe targeting the target region shown in Seq ID No. 72.
  • Seq ID No. 208 and Seq ID No. 209 are hypomethylated probes that target the target region shown in Seq ID No. 73, and Seq ID No. 348 and Seq ID No. 349 are both targeted to Seq ID No.
  • Seq ID No. 210 is a hypomethylated probe targeting the target region shown in Seq ID No. 74, and Seq ID No.
  • Seq ID No. 350 is a hypermethylated probe targeting the target region shown in Seq ID No. 74.
  • Seq ID No. 211 is a hypomethylated probe targeting the target region shown in Seq ID No. 75
  • Seq ID No. 351 is a hypermethylated probe targeting the target region shown in Seq ID No. 75.
  • Seq ID No. 212 is a hypomethylated probe targeting the target region shown in Seq ID No. 76
  • Seq ID No. 352 is a hypermethylated probe targeting the target region shown in Seq ID No. 76.
  • Seq ID No. 213 is a hypomethylated probe targeting the target region shown in Seq ID No. 77
  • Seq ID No. 353 is a hypermethylated probe targeting the target region shown in Seq ID No. 77.
  • Seq ID No. 214 is a hypomethylated probe that targets the target region shown in Seq ID No. 78
  • Seq ID No. 354 is a hypermethylated probe that targets the target region shown in Seq ID No. 78.
  • Seq ID No. 215 is a hypomethylated probe targeting the target region shown in Seq ID No. 79
  • Seq ID No. 355 is a hypermethylated probe targeting the target region shown in Seq ID No. 79.
  • Seq ID No.216 is a hypomethylated probe targeting the target region shown in Seq ID No.80
  • Seq ID No.356 is a hypermethylated probe targeting the target region shown in Seq ID No.80.
  • Seq ID No. 217 is a hypomethylated probe targeting the target region shown in Seq ID No. 81
  • Seq ID No. 357 is a hypermethylated probe targeting the target region shown in Seq ID No. 81.
  • Seq ID No. 218 is a hypomethylated probe targeting the target region shown in Seq ID No. 82
  • Seq ID No. 358 is a hypermethylated probe targeting the target region shown in Seq ID No. 82.
  • Seq ID No. 219 is a hypomethylated probe targeting the target region shown in Seq ID No. 83
  • Seq ID No. 359 is a hypermethylated probe targeting the target region shown in Seq ID No. 83.
  • Seq ID No. 220 is a hypomethylated probe targeting the target region shown in Seq ID No.
  • Seq ID No. 84 is a hypermethylated probe targeting the target region shown in Seq ID No. 84.
  • Seq ID No. 221 is a hypomethylated probe targeting the target region shown in Seq ID No. 85
  • Seq ID No. 361 is a hypermethylated probe targeting the target region shown in Seq ID No. 85.
  • Seq ID No. 222 is a hypomethylated probe targeting the target region shown in Seq ID No. 86
  • Seq ID No. 362 is a hypermethylated probe targeting the target region shown in Seq ID No. 86.
  • Seq ID No. 223 is a hypomethylated probe that targets the target region shown in Seq ID No. 87, and Seq ID No.
  • Seq ID No. 363 is a hypermethylated probe that targets the target region shown in Seq ID No. 87.
  • Seq ID No. 224 is a hypomethylated probe targeting the target region shown in Seq ID No. 88
  • Seq ID No. 364 is a hypermethylated probe targeting the target region shown in Seq ID No. 88.
  • Seq ID No. 225 and Seq ID No. 226 are both hypomethylated probes that target the target region shown in Seq ID No. 89
  • Seq ID No. 365 and Seq ID No. 366 are both targeted to Seq ID No.
  • Seq ID No. 227 is a hypomethylated probe that targets the target region shown in Seq ID No. 90, and Seq ID No.
  • Seq ID No. 367 is a hypermethylated probe that targets the target region shown in Seq ID No. 90.
  • Seq ID No. 228 is a hypomethylated probe that targets the target region shown in Seq ID No. 91
  • Seq ID No. 368 is a hypermethylated probe that targets the target region shown in Seq ID No. 91.
  • Seq ID No. 229 is a hypomethylated probe targeting the target region shown in Seq ID No. 92
  • Seq ID No. 369 is a hypermethylated probe targeting the target region shown in Seq ID No. 92.
  • Seq ID No. 230, Seq ID No. 231, and Seq ID No. 232 are all hypomethylated probes that target the target region shown in Seq ID No. 93, Seq ID No.
  • Seq ID No. 372 are all hypermethylated probes that target the target region shown in Seq ID No. 93.
  • Seq ID No. 233 is a hypomethylated probe targeting the target region shown in Seq ID No. 94
  • Seq ID No. 373 is a hypermethylated probe targeting the target region shown in Seq ID No. 94.
  • Seq ID No. 234 is a hypomethylated probe targeting the target region shown in Seq ID No. 95
  • Seq ID No. 374 is a hypermethylated probe targeting the target region shown in Seq ID No. 95.
  • Seq ID No. 235 is a hypomethylated probe targeting the target region shown in Seq ID No.
  • Seq ID No. 96 and Seq ID No. 375 is a hypermethylated probe targeting the target region shown in Seq ID No. 96.
  • Seq ID No. 236 and Seq ID No. 237 are hypomethylated probes that target the target region shown in Seq ID No. 97, and Seq ID No. 376 and Seq ID No. 377 are both targeted to Seq ID No. A hypermethylated probe in the target region shown in .97.
  • Seq ID No. 238 is a hypomethylated probe targeting the target region shown in Seq ID No. 98, and Seq ID No. 378 is a hypermethylated probe targeting the target region shown in Seq ID No. 98.
  • Seq ID No. 239 is a hypomethylated probe targeting the target region shown in Seq ID No.
  • Seq ID No. 379 is a hypermethylated probe targeting the target region shown in Seq ID No. 99.
  • Seq ID No. 240 is a hypomethylated probe targeting the target region shown in Seq ID No. 100
  • Seq ID No. 380 is a hypermethylated probe targeting the target region shown in Seq ID No. 100.
  • 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.
  • This article also provides a kit.
  • the kit includes the probe composition according to any one of the above embodiments.
  • the above-mentioned probe composition is used to prepare a kit for detecting esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer.
  • This article also provides a chip.
  • the probe composition according to any one of the above embodiments is immobilized on the chip.
  • This article also provides a method for detecting six cancers, including esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, by using the probe composition.
  • the detection method uses hybrid capture to enrich cfDNA, and uses NGS technology to detect methylation sites that are highly related to cancer. Covering the six malignant tumors with the highest incidence in my country (esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer and pancreatic 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 above six cancers by using the kit.
  • this application relates to six 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 65-100.
  • the hypomethylation probes include probes targeting cancer-specific regions Seq ID No.: any of 101-197, and probes targeting pan-cancer specific regions Seq ID No.: 198-199 Any, and Seq ID No. of probes targeting tissue-specific regions: Any of 200-240.
  • the hypermethylation probes include probes targeting cancer-specific regions, Seq ID No.: 241-337, and probes targeting pan-cancer specific regions, Seq ID No.: 338-339, Seq ID No.: any of 340-380 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.
  • Component volume Pre-amplified product 750ng corresponding volume Hyb human blocker 5 ⁇ l Junction blocker 6 ⁇ l Enhancer 5 ⁇ l
  • 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.
  • 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
  • Seq ID No. 75, Seq ID No. 76, Seq ID No. 84, Seq ID No. 85, Seq ID No. 86 and Seq ID No. 87 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.
  • Seq ID No. 64, Seq ID No. 78, Seq ID No. 79 , Seq ID No. 80, Seq ID No. 81 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).
  • Seq ID No. 70, Seq ID No. 73, Seq ID No. 82, and Seq ID No. 83 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, The lung tissue-specific markers are the most, and 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 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 22 As shown (Table 22 shows the detected target regions greater than or equal to the methylation threshold).
  • Seq ID No. 72 that are specific to liver tissue are greater than or equal to the respective thresholds of the methylation level, and there is no other tissue
  • 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, and Seq ID No. 74 are greater than or equal to their respective methylation levels. Threshold, 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, the esophageal tissue-specific markers are the most, and the final judgment is The sample is 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).
  • Seq ID No. 95, Seq ID No. 96, Seq ID No. 97, and Seq ID that are specific to pancreatic tissue No. 98 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, The pancreatic tissue-specific markers are the most, and the sample is finally judged to be a sample with pancreatic 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 probe composition screens 5.6 Kbp of a capture region, which capture region comprises 52 methylation change genes and non-coding DNA regions highly related to cancers. The probe composition can be used to detect methylation level changes of 6 cancers in one step: esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer and pancreatic cancer; and can be used for the early screening of an asymptomatic population and the prognostic detection of cancer patients.

Description

一种检测6种中国高发癌症的探针组合物A probe composition for detecting 6 high-incidence cancers in China 技术领域Technical field
本文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、肝癌和胰腺癌等6种肿瘤游离DNA甲基化水平变化的应用。This article relates to a cancer gene methylation detection composition, in particular to a probe composition that specifically recognizes the DNA sequence after Bisulfite treatment, and the detection of esophagus based on the high-throughput sequencing (NGS) method Application of the changes in the free DNA methylation level of 6 tumors including cancer, stomach cancer, colorectal cancer, lung cancer, liver cancer and pancreatic 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 variation 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26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 26 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 is free and extracellular in circulating blood. 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，本文提供了一种探针组合物，可用于食管癌、胃癌、结直肠癌、肺癌、肝癌和胰腺癌等6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 article provides a probe composition that can be used for the early stage of six cancers including esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer and pancreatic cancer. Screening. 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，本文涉及如下内容：Specifically, this article 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 癌和胰腺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: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, wherein the cancer-specific region is selected From Seq ID No.: Any of 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: a probe targeting a pan-cancer specific region, the pan-cancer specific region selected from any of Seq ID No.: 63-64.
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的探针，所述组织特异性区域选自Seq ID No.：65-100中的任意。3. The probe composition according to item 1, further comprising: a probe targeting a tissue-specific region, and the tissue-specific region is selected from any of Seq ID No.: 65-100.
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、74-76、84-89、99-100是食管的组织特异性靶区域。4. The probe composition according to item 3, characterized in that Seq ID No.: 66-67, 74-76, 84-89, 99-100 in the tissue-specific region are tissue-specific of the esophagus Sexual target area.
5.根据第3项所述的探针组合物，其特征在于，在所述组织特异性区域中，Seq ID No.：75、76、84-89是胃的组织特异性靶区域。5. The probe composition according to item 3, characterized in that, in the tissue-specific region, Seq ID No.: 75, 76, 84-89 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，在所述组织特异性区域中，Seq ID No.：77-81、93是结直肠的组织特异性靶区域。6. The probe composition according to item 3, characterized in that, in the tissue-specific region, Seq ID No.: 77-81, 93 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，在所述组织特异性区域中，Seq ID No.：65、68-70、73、82-83、90-91是肺的组织特异性靶区域。7. The probe composition according to item 3, characterized in that, in the tissue-specific region, Seq ID No.: 65, 68-70, 73, 82-83, 90-91 are pulmonary Tissue specific target area.
8.根据第3项所述的探针组合物，其特征在于，在所述组织特异性区域中，Seq ID No.：71-72、92是肝的组织特异性靶区域。8. The probe composition according to item 3, characterized in that, in the tissue-specific region, Seq ID No.: 71-72, 92 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，在所述组织特异性区域中，Seq ID No.：94-98是胰腺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.: 94-98 is a tissue-specific target region of the pancreas.
10.根据第1-9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。10. The probe composition according to any one of items 1-9, which comprises: a hypomethylation probe, which is specific to the bisulfite-converted cancer without CG methylation Hybridization of sexual regions, pan-cancer-specific regions, and tissue-specific regions, and; hypermethylation probes, which are all methylated with bisulfite-converted CG, the cancer-specific regions and pan-cancer-specific regions , And tissue-specific region hybridization.
11.根据第10项所述的探针组合物，其中所述探针组合物中的每一个探针长度为40～60bp。11. The probe composition according to item 10, wherein each probe in the probe composition is 40-60 bp in length.
12.根据第11项所述的探针组合物，其中所述探针组合物中的每一个探针长度为45～56bp，优选50～56bp，进一步优选50bp。12. The probe composition according to item 11, wherein the length of each probe in the probe composition is 45-56 bp, preferably 50-56 bp, and more preferably 50 bp.
13.根据第10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.：101-197中的任意，靶向泛癌特异性区域的探针Seq ID No.：198-199中的任意，和靶向组织特异性区域的 探针Seq ID No.：200-240中的任意。13. The probe composition according to item 10, wherein the hypomethylation probe includes a probe targeting a cancer-specific region. Seq ID No.: any of 101-197, targeting Seq ID No. of probes for pan-cancer specific regions: any of 198-199, and Seq ID No. of probes for tissue-specific regions: any of 200-240.
14.根据第10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.：241-337中的任意，靶向泛癌特异性区域的探针Seq ID No.：338-339中的任意，和靶向组织特异性区域的探针Seq ID No.：340-380中的任意。14. The probe composition according to item 10, wherein the hypermethylation probe includes a probe targeting a cancer-specific region Seq ID No.: any of 241-337, targeting pan-cancer Seq ID No. of probes for specific regions: any of 338-339, and Seq ID No. of probes for tissue-specific regions: any of 340-380.
15.一种试剂盒，其包括第1-14项中任一项所述的探针组合物。15. A kit comprising the probe composition according to any one of items 1-14.
16.一种第1-14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、肝癌和胰腺癌的试剂盒中的应用。16. An application of the probe composition according to any one of items 1-14 in the preparation of a kit for detecting esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer and pancreatic cancer.
17.一种芯片，其上固定有包括第1-14项中任一项所述的探针组合物。17. A chip on which the probe composition comprising any one of items 1-14 is immobilized.
本文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、肝癌和胰腺癌等6种癌症的方法。This article also provides a method for detecting six cancers, including esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, by using the probe composition.
本文同时也提供了一种利用所述试剂盒同时检测上述6种癌症的甲基化水平改变的方法。This article also provides a method for simultaneously detecting changes in the methylation level of the above 6 cancers by using the kit.
本申请同时也提供了一种利用所述试剂盒同时检测上述6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 above six cancers 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6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 detect the methylation level changes of 6 common cancers in China at the same time, 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Panel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 enterprises, one Panel can complete the screening of major cancers, which saves the cost of probe synthesis, 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为本文的实施操作流程Figure 1 shows the implementation process of this article
具体实施方式Detailed ways
本文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、肝癌和胰腺癌等6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6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 article provides a probe composition for cancer gene methylation detection. Based on the high-throughput sequencing (NGS) method to detect the changes in the free DNA methylation level of 6 tumors including esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer and pancreatic cancer. It can simultaneously detect the methylation level changes of 6 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文所属领域的普通技术人员通常理解的含义。Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which this document 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。在本文中，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 context, 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.
以下详细描述本文的技术方案。The technical solution of this article is described in detail below.
在本文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、肝癌和胰腺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的探针，所述组织特异性区域选自Seq ID No.：65-100中的任意。在所述组织特异性区域中，Seq ID No.：66-67、74-76、84-89、99-100是食管的组织特异性靶区域。In a specific embodiment herein, a probe composition includes: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, wherein, The cancer-specific region is selected from any of Seq ID No.: 1-62; a probe targeting a pan-cancer specific region, and the pan-cancer specific region is selected from Seq ID No.: 63-64 Any; and a probe targeting a tissue-specific region, the tissue-specific region is selected from any of Seq ID No.: 65-100. Among the tissue-specific regions, Seq ID No.: 66-67, 74-76, 84-89, and 99-100 are tissue-specific target regions of the esophagus.
在本文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、肝癌和胰腺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的探针，所述组织特异性区域选自Seq ID No.：65-100中的任意。在所述组织特异性区域中，Seq ID No.：75、76、84-89是胃的组织特异性靶区域。In a specific embodiment herein, a probe composition includes: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, wherein, The cancer-specific region is selected from any of Seq ID No.: 1-62; a probe targeting a pan-cancer specific region, and the pan-cancer specific region is selected from Seq ID No.: 63-64 Any; and a probe targeting a tissue-specific region, the tissue-specific region is selected from any of Seq ID No.: 65-100. Among the tissue-specific regions, Seq ID No.: 75, 76, 84-89 are tissue-specific target regions of the stomach.
在本文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、肝癌和胰腺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的探针，所述组织特异性区域选自Seq ID No.：65-100中的任意。在所述组织特异性区域中，Seq ID No.：77-81、93是结直肠的组织特异性靶区域。In a specific embodiment herein, a probe composition includes: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, wherein, The cancer-specific region is selected from any of Seq ID No.: 1-62; a probe targeting a pan-cancer specific region, and the pan-cancer specific region is selected from Seq ID No.: 63-64 Any; and a probe targeting a tissue-specific region, the tissue-specific region is selected from any of Seq ID No.: 65-100. Among the tissue-specific regions, Seq ID No.: 77-81 and 93 are tissue-specific target regions of the colorectal.
在本文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、肝癌和胰腺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的探针，所述组织特异性区域选自Seq ID No.：65-100中的任意。在所述组织特异性区域中，Seq ID No.：65、68-70、73、82-83、90-91是肺的组织特异性靶区域。In a specific embodiment herein, a probe composition includes: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, wherein, The cancer-specific region is selected from any of Seq ID No.: 1-62; a probe targeting a pan-cancer specific region, and the pan-cancer specific region is selected from Seq ID No.: 63-64 Any; and a probe targeting a tissue-specific region, the tissue-specific region is selected from any of Seq ID No.: 65-100. Among the tissue-specific regions, Seq ID No.: 65, 68-70, 73, 82-83, and 90-91 are tissue-specific target regions of the lung.
在本文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、肝癌和胰腺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的探针，所述组织特异性区域选自Seq ID No.：65-100中的任意。在所述组织特异性区域中，Seq ID No.：71-72、92是肝的组织特异性靶区域。In a specific embodiment herein, a probe composition includes: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, wherein, The cancer-specific region is selected from any of Seq ID No.: 1-62; a probe targeting a pan-cancer specific region, and the pan-cancer specific region is selected from Seq ID No.: 63-64 Any; and a probe targeting a tissue-specific region, the tissue-specific region is selected from any of Seq ID No.: 65-100. Among the tissue-specific regions, Seq ID No.: 71-72, 92 are tissue-specific target regions of the liver.
在本文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、肝癌和胰腺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的探针，所述组织特异性区域选自Seq ID No.：65-100中的任意。在所述组织特异性区域中，Seq ID No.：94-98是胰腺的组织特异性靶区域。In a specific embodiment herein, a probe composition includes: a probe targeting a specific region of any cancer among esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, wherein, The cancer-specific region is selected from any of Seq ID No.: 1-62; a probe targeting a pan-cancer specific region, and the pan-cancer specific region is selected from Seq ID No.: 63-64 Any; and a probe targeting a tissue-specific region, the tissue-specific region is selected from any of Seq ID No.: 65-100. Among the tissue-specific regions, Seq ID No.: 94-98 is the tissue-specific target region of the pancreas.
在本文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。所述低甲基化探针包括靶向癌症特异性区域的探针Seq ID No.：101-197中的任意，靶向泛癌特异性区域的探针Seq ID No.：198-199中的任意，和靶向组织特异性区域的探针Seq ID No.：200-240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.：241-337中的任意，靶向泛癌特异性区域的探针Seq ID No.：338-339中的任意，和靶向组织特异性区域的探针Seq ID No.：340-380中的任意。In a specific embodiment herein, the above-mentioned probe composition includes a hypomethylation probe, which is different from the cancer-specific region and pan-cancer-specific region converted by bisulfite without CG methylation. Hybridization of sexual regions and tissue-specific regions, and hypermethylation probes that hybridize to the cancer-specific regions, pan-cancer-specific regions, and tissue-specific regions where bisulfite-converted CG is fully methylated . The hypomethylation probes include probes targeting cancer-specific regions Seq ID No.: any of 101-197, and probes targeting pan-cancer specific regions Seq ID No.: 198-199 Any, and Seq ID No. of probes targeting tissue-specific regions: Any of 200-240. The hypermethylation probes include probes targeting cancer-specific regions, Seq ID No.: 241-337, and probes targeting pan-cancer specific regions, Seq ID No.: 338-339, Seq ID No.: any of 340-380 probes targeting tissue-specific regions.
如下表1所示，其中Seq ID No.101、Seq ID No.102和Seq ID No.103均是靶向Seq ID No.1所示靶区域的低甲基化探针，Seq ID No.241、Seq ID No.242和Seq ID No.243均是靶向Seq ID No.1所示靶区域的高甲基化探针。Seq ID No.104和Seq ID No.105均是靶向Seq ID No.2所示靶区域的低甲基化探针，Seq ID No.244和Seq ID No.245均是靶向Seq ID No.2所示靶区域的高甲基化探针。Seq ID No.106和Seq ID No.107均是靶向Seq ID No.3所示靶区域的低甲基化探针，Seq ID No.246和Seq ID No.247均是靶向Seq ID  No.3所示靶区域的高甲基化探针。Seq ID No.108是靶向Seq ID No.4所示靶区域的低甲基化探针，Seq ID No.248是靶向Seq ID No.4所示靶区域的高甲基化探针。Seq ID No.109和Seq ID No.110均是靶向Seq ID No.5所示靶区域的低甲基化探针，Seq ID No.249和Seq ID No.250均是靶向Seq ID No.3所示靶区域的高甲基化探针。Seq ID No.111是靶向Seq ID No.6所示靶区域的低甲基化探针，Seq ID No.251是靶向Seq ID No.6所示靶区域的高甲基化探针。Seq ID No.112是靶向Seq ID No.7所示靶区域的低甲基化探针，Seq ID No.252是靶向Seq ID No.7所示靶区域的高甲基化探针。Seq ID No.113和Seq ID No.114均是靶向Seq ID No.8所示靶区域的低甲基化探针，Seq ID No.253和Seq ID No.254均是靶向Seq ID No.8所示靶区域的高甲基化探针。Seq ID No.115、Seq ID No.116和Seq ID No.117均是靶向Seq ID No.9所示靶区域的低甲基化探针，Seq ID No.255、Seq ID No.256和Seq ID No.257均是靶向Seq ID No.9所示靶区域的高甲基化探针。Seq ID No.118是靶向Seq ID No.10所示靶区域的低甲基化探针，Seq ID No.258是靶向Seq ID No.10所示靶区域的高甲基化探针。Seq ID No.119是靶向Seq ID No.11所示靶区域的低甲基化探针，Seq ID No.259是靶向Seq ID No.11所示靶区域的高甲基化探针。Seq ID No.120是靶向Seq ID No.12所示靶区域的低甲基化探针，Seq ID No.260是靶向Seq ID No.12所示靶区域的高甲基化探针。Seq ID No.121是靶向Seq ID No.13所示靶区域的低甲基化探针，Seq ID No.261是靶向Seq ID No.13所示靶区域的高甲基化探针。Seq ID No.122和Seq ID No.123均是靶向Seq ID No.14所示靶区域的低甲基化探针，Seq ID No.262和Seq ID No.263均是靶向Seq ID No.14所示靶区域的高甲基化探针。Seq ID No.124和Seq ID No.125均是靶向Seq ID No.15所示靶区域的低甲基化探针，Seq ID No.264和Seq ID No.265均是靶向Seq ID No.15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.16所示靶区域的低甲基化探针，Seq ID No.266和Seq ID No.267均是靶向Seq ID No.16所示靶区域的高甲基化探针。Seq ID No.128和Seq ID No.129均是靶向Seq ID No.17所示靶区域的低甲基化探针，Seq ID No.268和Seq ID No.269均是靶向Seq ID No.17所示靶区域的高甲基化探针。Seq ID No.130是靶向Seq ID No.18所示靶区域的低甲基化探针，Seq ID No.270是靶向Seq ID No.18所示靶区域的高甲基化探针。 Seq ID No.131和Seq ID No.132均是靶向Seq ID No.19所示靶区域的低甲基化探针，Seq ID No.271和Seq ID No.272均是靶向Seq ID No.19所示靶区域的高甲基化探针。Seq ID No.133是靶向Seq ID No.20所示靶区域的低甲基化探针，Seq ID No.273是靶向Seq ID No.20所示靶区域的高甲基化探针。Seq ID No.134是靶向Seq ID No.21所示靶区域的低甲基化探针，Seq ID No.274是靶向Seq ID No.21所示靶区域的高甲基化探针。Seq ID No.135是靶向Seq ID No.22所示靶区域的低甲基化探针，Seq ID No.275是靶向Seq ID No.22所示靶区域的高甲基化探针。Seq ID No.136、Seq ID No.137和Seq ID No.138均是靶向Seq ID No.23所示靶区域的低甲基化探针，Seq ID No.276、Seq ID No.277和Seq ID No.278均是靶向Seq ID No.23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.24所示靶区域的低甲基化探针，Seq ID No.279和Seq ID No.280均是靶向Seq ID No.24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.25所示靶区域的低甲基化探针，Seq ID No.281和Seq ID No.282均是靶向Seq ID No.25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.26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.26所示靶区域的高甲基化探针。Seq ID No.145是靶向Seq ID No.27所示靶区域的低甲基化探针，Seq ID No.285是靶向Seq ID No.27所示靶区域的高甲基化探针。Seq ID No.146和Seq ID No.147均是靶向Seq ID No.28所示靶区域的低甲基化探针，Seq ID No.286和Seq ID No.287均是靶向Seq ID No.28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.29所示靶区域的低甲基化探针，Seq ID No.288和Seq ID No.289均是靶向Seq ID No.29所示靶区域的高甲基化探针。Seq ID No.150是靶向Seq ID No.30所示靶区域的低甲基化探针，Seq ID No.290是靶向Seq ID No.30所示靶区域的高甲基化探针。Seq ID No.151和Seq ID No.152均是靶向Seq ID No.31所示靶区域的低甲基化探针，Seq ID No.291和Seq ID No.292均是靶向Seq ID No.31所示靶区域的高甲基化探针。Seq ID No.153是靶向Seq ID No.32所示靶区域的低甲基化探针，Seq ID No.293是靶向Seq ID No.32所示靶区域的高甲基化探针。Seq ID No.154和Seq ID No.155均是靶向Seq ID No.33所示靶区域的低甲基化探针，Seq ID  No.294和Seq ID No.295均是靶向Seq ID No.33所示靶区域的高甲基化探针。Seq ID No.156是靶向Seq ID No.34所示靶区域的低甲基化探针，Seq ID No.296是靶向Seq ID No.34所示靶区域的高甲基化探针。Seq ID No.157和Seq ID No.158均是靶向Seq ID No.35所示靶区域的低甲基化探针，Seq ID No.297和Seq ID No.298均是靶向Seq ID No.35所示靶区域的高甲基化探针。Seq ID No.159是靶向Seq ID No.36所示靶区域的低甲基化探针，Seq ID No.299是靶向Seq ID No.36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.37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.37所示靶区域的高甲基化探针。Seq ID No.162和Seq ID No.163均是靶向Seq ID No.38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.38所示靶区域的高甲基化探针。Seq ID No.164是靶向Seq ID No.39所示靶区域的低甲基化探针，Seq ID No.304是靶向Seq ID No.39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.40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.40所示靶区域的高甲基化探针。Seq ID No.167是靶向Seq ID No.41所示靶区域的低甲基化探针，Seq ID No.307是靶向Seq ID No.41所示靶区域的高甲基化探针。Seq ID No.168是靶向Seq ID No.42所示靶区域的低甲基化探针，Seq ID No.308是靶向Seq ID No.42所示靶区域的高甲基化探针。Seq ID No.169是靶向Seq ID No.43所示靶区域的低甲基化探针，Seq ID No.309是靶向Seq ID No.43所示靶区域的高甲基化探针。Seq ID No.170是靶向Seq ID No.44所示靶区域的低甲基化探针，Seq ID No.310是靶向Seq ID No.44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.45所示靶区域的低甲基化探针，Seq ID No.311和Seq ID No.312均是靶向Seq ID No.45所示靶区域的高甲基化探针。Seq ID No.173是靶向Seq ID No.46所示靶区域的低甲基化探针，Seq ID No.313是靶向Seq ID No.46所示靶区域的高甲基化探针。Seq ID No.174是靶向Seq ID No.47所示靶区域的低甲基化探针，Seq ID No.314是靶向Seq ID No.47所示靶区域的高甲基化探针。Seq ID No.175是靶向Seq ID No.48所示靶区域的低甲基化探针，Seq ID No.315是靶向Seq ID No.48所示靶区域的高甲基化探针。Seq ID No.176是靶向Seq  ID No.49所示靶区域的低甲基化探针，Seq ID No.316是靶向Seq ID No.49所示靶区域的高甲基化探针。Seq ID No.177是靶向Seq ID No.50所示靶区域的低甲基化探针，Seq ID No.317是靶向Seq ID No.50所示靶区域的高甲基化探针。Seq ID No.178和Seq ID No.179均是靶向Seq ID No.51所示靶区域的低甲基化探针，Seq ID No.318和Seq ID No.319均是靶向Seq ID No.51所示靶区域的高甲基化探针。Seq ID No.180是靶向Seq ID No.52所示靶区域的低甲基化探针，Seq ID No.320是靶向Seq ID No.52所示靶区域的高甲基化探针。Seq ID No.181和Seq ID No.182均是靶向Seq ID No.53所示靶区域的低甲基化探针，Seq ID No.321和Seq ID No.322均是靶向Seq ID No.53所示靶区域的高甲基化探针。Seq ID No.183是靶向Seq ID No.54所示靶区域的低甲基化探针，Seq ID No.323是靶向Seq ID No.54所示靶区域的高甲基化探针。Seq ID No.184和Seq ID No.185均是靶向Seq ID No.55所示靶区域的低甲基化探针，Seq ID No.324和Seq ID No.325均是靶向Seq ID No.55所示靶区域的高甲基化探针。Seq ID No.186和Seq ID No.187均是靶向Seq ID No.56所示靶区域的低甲基化探针，Seq ID No.326和Seq ID No.327均是靶向Seq ID No.56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.57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.57所示靶区域的高甲基化探针。Seq ID No.190和Seq ID No.191均是靶向Seq ID No.58所示靶区域的低甲基化探针，Seq ID No.330和Seq ID No.331均是靶向Seq ID No.58所示靶区域的高甲基化探针。Seq ID No.192和Seq ID No.193均是靶向Seq ID No.59所示靶区域的低甲基化探针，Seq ID No.332和Seq ID No.333均是靶向Seq ID No.59所示靶区域的高甲基化探针。Seq ID No.194是靶向Seq ID No.60所示靶区域的低甲基化探针，Seq ID No.334是靶向Seq ID No.60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.61所示靶区域的低甲基化探针，Seq ID No.335和Seq ID No.336均是靶向Seq ID No.61所示靶区域的高甲基化探针。Seq ID No.197是靶向Seq ID No.62所示靶区域的低甲基化探针，Seq ID No.337是靶向Seq ID No.62所示靶区域的高甲基化探针。Seq ID No.198是靶向Seq ID No.63所示靶区域的低甲基化探针，Seq ID No.338是靶向Seq ID No.63所示靶区域的高甲基化探针。Seq ID  No.199是靶向Seq ID No.64所示靶区域的低甲基化探针，Seq ID No.339是靶向Seq ID No.64所示靶区域的高甲基化探针。Seq ID No.200是靶向Seq ID No.65所示靶区域的低甲基化探针，Seq ID No.340是靶向Seq ID No.65所示靶区域的高甲基化探针。Seq ID No.201是靶向Seq ID No.66所示靶区域的低甲基化探针，Seq ID No.341是靶向Seq ID No.66所示靶区域的高甲基化探针。Seq ID No.202是靶向Seq ID No.67所示靶区域的低甲基化探针，Seq ID No.342是靶向Seq ID No.67所示靶区域的高甲基化探针。Seq ID No.203是靶向Seq ID No.68所示靶区域的低甲基化探针，Seq ID No.343是靶向Seq ID No.68所示靶区域的高甲基化探针。Seq ID No.204是靶向Seq ID No.69所示靶区域的低甲基化探针，Seq ID No.344是靶向Seq ID No.69所示靶区域的高甲基化探针。Seq ID No.205是靶向Seq ID No.70所示靶区域的低甲基化探针，Seq ID No.345是靶向Seq ID No.70所示靶区域的高甲基化探针。Seq ID No.206是靶向Seq ID No.71所示靶区域的低甲基化探针，Seq ID No.346是靶向Seq ID No.71所示靶区域的高甲基化探针。Seq ID No.207是靶向Seq ID No.72所示靶区域的低甲基化探针，Seq ID No.347是靶向Seq ID No.72所示靶区域的高甲基化探针。Seq ID No.208和Seq ID No.209均是靶向Seq ID No.73所示靶区域的低甲基化探针，Seq ID No.348和Seq ID No.349均是靶向Seq ID No.73所示靶区域的高甲基化探针。Seq ID No.210是靶向Seq ID No.74所示靶区域的低甲基化探针，Seq ID No.350是靶向Seq ID No.74所示靶区域的高甲基化探针。Seq ID No.211是靶向Seq ID No.75所示靶区域的低甲基化探针，Seq ID No.351是靶向Seq ID No.75所示靶区域的高甲基化探针。Seq ID No.212是靶向Seq ID No.76所示靶区域的低甲基化探针，Seq ID No.352是靶向Seq ID No.76所示靶区域的高甲基化探针。Seq ID No.213是靶向Seq ID No.77所示靶区域的低甲基化探针，Seq ID No.353是靶向Seq ID No.77所示靶区域的高甲基化探针。Seq ID No.214是靶向Seq ID No.78所示靶区域的低甲基化探针，Seq ID No.354是靶向Seq ID No.78所示靶区域的高甲基化探针。Seq ID No.215是靶向Seq ID No.79所示靶区域的低甲基化探针，Seq ID No.355是靶向Seq ID No.79所示靶区域的高甲基化探针。Seq ID No.216是靶向Seq ID No.80所示靶区域的低甲基化探针，Seq ID No.356是靶向Seq ID No.80所示靶区域的高甲基化探针。Seq ID  No.217是靶向Seq ID No.81所示靶区域的低甲基化探针，Seq ID No.357是靶向Seq ID No.81所示靶区域的高甲基化探针。Seq ID No.218是靶向Seq ID No.82所示靶区域的低甲基化探针，Seq ID No.358是靶向Seq ID No.82所示靶区域的高甲基化探针。Seq ID No.219是靶向Seq ID No.83所示靶区域的低甲基化探针，Seq ID No.359是靶向Seq ID No.83所示靶区域的高甲基化探针。Seq ID No.220是靶向Seq ID No.84所示靶区域的低甲基化探针，Seq ID No.360是靶向Seq ID No.84所示靶区域的高甲基化探针。Seq ID No.221是靶向Seq ID No.85所示靶区域的低甲基化探针，Seq ID No.361是靶向Seq ID No.85所示靶区域的高甲基化探针。Seq ID No.222是靶向Seq ID No.86所示靶区域的低甲基化探针，Seq ID No.362是靶向Seq ID No.86所示靶区域的高甲基化探针。Seq ID No.223是靶向Seq ID No.87所示靶区域的低甲基化探针，Seq ID No.363是靶向Seq ID No.87所示靶区域的高甲基化探针。Seq ID No.224是靶向Seq ID No.88所示靶区域的低甲基化探针，Seq ID No.364是靶向Seq ID No.88所示靶区域的高甲基化探针。Seq ID No.225和Seq ID No.226均是靶向Seq ID No.89所示靶区域的低甲基化探针，Seq ID No.365和Seq ID No.366均是靶向Seq ID No.89所示靶区域的高甲基化探针。Seq ID No.227是靶向Seq ID No.90所示靶区域的低甲基化探针，Seq ID No.367是靶向Seq ID No.90所示靶区域的高甲基化探针。Seq ID No.228是靶向Seq ID No.91所示靶区域的低甲基化探针，Seq ID No.368是靶向Seq ID No.91所示靶区域的高甲基化探针。Seq ID No.229是靶向Seq ID No.92所示靶区域的低甲基化探针，Seq ID No.369是靶向Seq ID No.92所示靶区域的高甲基化探针。Seq ID No.230、Seq ID No.231和Seq ID No.232均是靶向Seq ID No.93所示靶区域的低甲基化探针，Seq ID No.370、Seq ID No.371和Seq ID No.372均是靶向Seq ID No.93所示靶区域的高甲基化探针。Seq ID No.233是靶向Seq ID No.94所示靶区域的低甲基化探针，Seq ID No.373是靶向Seq ID No.94所示靶区域的高甲基化探针。Seq ID No.234是靶向Seq ID No.95所示靶区域的低甲基化探针，Seq ID No.374是靶向Seq ID No.95所示靶区域的高甲基化探针。Seq ID No.235是靶向Seq ID No.96所示靶区域的低甲基化探针，Seq ID No.375是靶向Seq ID No.96所示靶区域的高甲基化探针。Seq ID No.236和Seq ID No.237均是靶向Seq ID No.97所示靶区域的低甲基 化探针，Seq ID No.376和Seq ID No.377均是靶向Seq ID No.97所示靶区域的高甲基化探针。Seq ID No.238是靶向Seq ID No.98所示靶区域的低甲基化探针，Seq ID No.378是靶向Seq ID No.98所示靶区域的高甲基化探针。Seq ID No.239是靶向Seq ID No.99所示靶区域的低甲基化探针，Seq ID No.379是靶向Seq ID No.99所示靶区域的高甲基化探针。Seq ID No.240是靶向Seq ID No.100所示靶区域的低甲基化探针，Seq ID No.380是靶向Seq ID No.100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. 101, Seq ID No. 102, and Seq ID No. 103 are all hypomethylated probes that target the target region shown in Seq ID No. 1, Seq ID No. 241 , Seq ID No. 242 and Seq ID No. 243 are hypermethylated probes that target the target region shown in Seq ID No. 1. Seq ID No. 104 and Seq ID No. 105 are hypomethylated probes that target the target region shown in Seq ID No. 2, and Seq ID No. 244 and Seq ID No. 245 are both targeted to Seq ID No. .2 Hypermethylated probes in the target region shown. Seq ID No. 106 and Seq ID No. 107 are hypomethylated probes that target the target region shown in Seq ID No. 3, and Seq ID No. 246 and Seq ID No. 247 are both targeted to Seq ID No. .3 Hypermethylated probes in the target region shown. Seq ID No. 108 is a hypomethylated probe targeting the target region shown in Seq ID No. 4, and Seq ID No. 248 is a hypermethylated probe targeting the target region shown in Seq ID No. 4. Seq ID No. 109 and Seq ID No. 110 are hypomethylated probes that target the target region shown in Seq ID No. 5, and Seq ID No. 249 and Seq ID No. 250 are both targeted to Seq ID No. .3 Hypermethylated probes in the target region shown. Seq ID No. 111 is a hypomethylated probe that targets the target region shown in Seq ID No. 6, and Seq ID No. 251 is a hypermethylated probe that targets the target region shown in Seq ID No. 6. Seq ID No. 112 is a hypomethylated probe targeting the target region shown in Seq ID No. 7, and Seq ID No. 252 is a hypermethylated probe targeting the target region shown in Seq ID No. 7. Seq ID No. 113 and Seq ID No. 114 are hypomethylated probes that target the target region shown in Seq ID No. 8, and Seq ID No. 253 and Seq ID No. 254 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .8. Seq ID No. 115, Seq ID No. 116, and Seq ID No. 117 are all hypomethylated probes that target the target region shown in Seq ID No. 9, Seq ID No. 255, Seq ID No. 256 and Seq ID No. 256. Seq ID No. 257 is a hypermethylated probe that targets the target region shown in Seq ID No. 9. Seq ID No.118 is a hypomethylated probe targeting the target region shown in Seq ID No.10, and Seq ID No.258 is a hypermethylated probe targeting the target region shown in Seq ID No.10. Seq ID No. 119 is a hypomethylated probe targeting the target region shown in Seq ID No. 11, and Seq ID No. 259 is a hypermethylated probe targeting the target region shown in Seq ID No. 11. Seq ID No. 120 is a hypomethylated probe targeting the target region shown in Seq ID No. 12, and Seq ID No. 260 is a hypermethylated probe targeting the target region shown in Seq ID No. 12. Seq ID No. 121 is a hypomethylated probe targeting the target region shown in Seq ID No. 13, and Seq ID No. 261 is a hypermethylated probe targeting the target region shown in Seq ID No. 13. Seq ID No. 122 and Seq ID No. 123 are hypomethylation probes that target the target region shown in Seq ID No. 14, and Seq ID No. 262 and Seq ID No. 263 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .14. Seq ID No. 124 and Seq ID No. 125 are hypomethylated probes that target the target region shown in Seq ID No. 15, and Seq ID No. 264 and Seq ID No. 265 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .15. Seq ID No. 126 and Seq ID No. 127 are both hypomethylated probes that target the target region shown in Seq ID No. 16, and Seq ID No. 266 and Seq ID No. 267 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .16. Seq ID No. 128 and Seq ID No. 129 are both hypomethylated probes that target the target region shown in Seq ID No. 17, and Seq ID No. 268 and Seq ID No. 269 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .17. Seq ID No. 130 is a hypomethylated probe targeting the target region shown in Seq ID No. 18, and Seq ID No. 270 is a hypermethylated probe targeting the target region shown in Seq ID No. 18. Seq ID No. 131 and Seq ID No. 132 are hypomethylated probes that target the target region shown in Seq ID No. 19, and Seq ID No. 271 and Seq ID No. 272 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .19. Seq ID No. 133 is a hypomethylated probe targeting the target region shown in Seq ID No. 20, and Seq ID No. 273 is a hypermethylated probe targeting the target region shown in Seq ID No. 20. Seq ID No. 134 is a hypomethylated probe targeting the target region shown in Seq ID No. 21, and Seq ID No. 274 is a hypermethylated probe targeting the target region shown in Seq ID No. 21. Seq ID No. 135 is a hypomethylated probe targeting the target region shown in Seq ID No. 22, and Seq ID No. 275 is a hypermethylated probe targeting the target region shown in Seq ID No. 22. Seq ID No. 136, Seq ID No. 137, and Seq ID No. 138 are hypomethylated probes that target the target region shown in Seq ID No. 23, Seq ID No. 276, Seq ID No. 277, and Seq ID No. 277. Seq ID No. 278 are all hypermethylated probes that target the target region shown in Seq ID No. 23. Seq ID No. 139 and Seq ID No. 140 are both hypomethylated probes that target the target region shown in Seq ID No. 24, and Seq ID No. 279 and Seq ID No. 280 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .24. Seq ID No. 141 and Seq ID No. 142 are hypomethylated probes that target the target region shown in Seq ID No. 25, and Seq ID No. 281 and Seq ID No. 282 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .25. Seq ID No. 143 and Seq ID No. 144 are hypomethylated probes that target the target region shown in Seq ID No. 26, and Seq ID No. 283 and Seq ID No. 284 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .26. Seq ID No. 145 is a hypomethylated probe targeting the target region shown in Seq ID No. 27, and Seq ID No. 285 is a hypermethylated probe targeting the target region shown in Seq ID No. 27. Seq ID No. 146 and Seq ID No. 147 are hypomethylated probes that target the target region shown in Seq ID No. 28, and Seq ID No. 286 and Seq ID No. 287 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .28. Seq ID No. 148 and Seq ID No. 149 are both hypomethylated probes that target the target region shown in Seq ID No. 29, and Seq ID No. 288 and Seq ID No. 289 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .29. Seq ID No. 150 is a hypomethylated probe that targets the target region shown in Seq ID No. 30, and Seq ID No. 290 is a hypermethylated probe that targets the target region shown in Seq ID No. 30. Seq ID No. 151 and Seq ID No. 152 are both hypomethylated probes that target the target region shown in Seq ID No. 31, and Seq ID No. 291 and Seq ID No. 292 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .31. Seq ID No. 153 is a hypomethylated probe targeting the target region shown in Seq ID No. 32, and Seq ID No. 293 is a hypermethylated probe targeting the target region shown in Seq ID No. 32. Seq ID No. 154 and Seq ID No. 155 are hypomethylated probes that target the target region shown in Seq ID No. 33, and Seq ID No. 294 and Seq ID No. 295 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .33. Seq ID No.156 is a hypomethylated probe targeting the target region shown in Seq ID No.34, and Seq ID No.296 is a hypermethylated probe targeting the target region shown in Seq ID No.34. Seq ID No. 157 and Seq ID No. 158 are hypomethylation probes that target the target region shown in Seq ID No. 35, and Seq ID No. 297 and Seq ID No. 298 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .35. Seq ID No. 159 is a hypomethylated probe targeting the target region shown in Seq ID No. 36, and Seq ID No. 299 is a hypermethylated probe targeting the target region shown in Seq ID No. 36. Seq ID No. 160 and Seq ID No. 161 are hypomethylated probes that target the target region shown in Seq ID No. 37, 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 .37. Seq ID No. 162 and Seq ID No. 163 are hypomethylation probes that target the target region shown in Seq ID No. 38, 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 .38. Seq ID No.164 is a hypomethylated probe targeting the target region shown in Seq ID No.39, and Seq ID No.304 is a hypermethylated probe targeting the target region shown in Seq ID No.39. Seq ID No. 165 and Seq ID No. 166 are hypomethylated probes that target the target region shown in Seq ID No. 40, 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 .40. Seq ID No. 167 is a hypomethylated probe targeting the target region shown in Seq ID No. 41, and Seq ID No. 307 is a hypermethylated probe targeting the target region shown in Seq ID No. 41. Seq ID No.168 is a hypomethylated probe targeting the target region shown in Seq ID No.42, and Seq ID No.308 is a hypermethylated probe targeting the target region shown in Seq ID No.42. Seq ID No. 169 is a hypomethylated probe targeting the target region shown in Seq ID No. 43, and Seq ID No. 309 is a hypermethylated probe targeting the target region shown in Seq ID No. 43. Seq ID No. 170 is a hypomethylated probe targeting the target region shown in Seq ID No. 44, and Seq ID No. 310 is a hypermethylated probe targeting the target region shown in Seq ID No. 44. Seq ID No. 171 and Seq ID No. 172 are both hypomethylated probes that target the target region shown in Seq ID No. 45, and Seq ID No. 311 and Seq ID No. 312 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .45. Seq ID No. 173 is a hypomethylated probe targeting the target region shown in Seq ID No. 46, and Seq ID No. 313 is a hypermethylated probe targeting the target region shown in Seq ID No. 46. Seq ID No. 174 is a hypomethylated probe that targets the target region shown in Seq ID No. 47, and Seq ID No. 314 is a hypermethylated probe that targets the target region shown in Seq ID No. 47. Seq ID No. 175 is a hypomethylated probe targeting the target region shown in Seq ID No. 48, and Seq ID No. 315 is a hypermethylated probe targeting the target region shown in Seq ID No. 48. Seq ID No. 176 is a hypomethylated probe that targets the target region shown in Seq ID No. 49, and Seq ID No. 316 is a hypermethylated probe that targets the target region shown in Seq ID No. 49. Seq ID No. 177 is a hypomethylated probe targeting the target region shown in Seq ID No. 50, and Seq ID No. 317 is a hypermethylated probe targeting the target region shown in Seq ID No. 50. Seq ID No. 178 and Seq ID No. 179 are both hypomethylated probes that target the target region shown in Seq ID No. 51, and Seq ID No. 318 and Seq ID No. 319 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .51. Seq ID No. 180 is a hypomethylated probe targeting the target region shown in Seq ID No. 52, and Seq ID No. 320 is a hypermethylated probe targeting the target region shown in Seq ID No. 52. Seq ID No. 181 and Seq ID No. 182 are hypomethylation probes that target the target region shown in Seq ID No. 53, and Seq ID No. 321 and Seq ID No. 322 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .53. Seq ID No. 183 is a hypomethylated probe targeting the target region shown in Seq ID No. 54, and Seq ID No. 323 is a hypermethylated probe targeting the target region shown in Seq ID No. 54. Seq ID No. 184 and Seq ID No. 185 are hypomethylated probes that target the target region shown in Seq ID No. 55, and Seq ID No. 324 and Seq ID No. 325 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .55. Seq ID No. 186 and Seq ID No. 187 are hypomethylated probes that target the target region shown in Seq ID No. 56, and Seq ID No. 326 and Seq ID No. 327 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .56. Seq ID No. 188 and Seq ID No. 189 are both hypomethylated probes that target the target region shown in Seq ID No. 57, and Seq ID No. 328 and Seq ID No. 329 are both targeted to Seq ID No. A hypermethylated probe in the target region shown in .57. Seq ID No. 190 and Seq ID No. 191 are hypomethylated probes that target the target region shown in Seq ID No. 58, and Seq ID No. 330 and Seq ID No. 331 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .58. Seq ID No. 192 and Seq ID No. 193 are both hypomethylated probes that target the target region shown in Seq ID No. 59, and Seq ID No. 332 and Seq ID No. 333 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .59. Seq ID No. 194 is a hypomethylated probe targeting the target region shown in Seq ID No. 60, and Seq ID No. 334 is a hypermethylated probe targeting the target region shown in Seq ID No. 60. Seq ID No. 195 and Seq ID No. 196 are hypomethylated probes that target the target region shown in Seq ID No. 61, and Seq ID No. 335 and Seq ID No. 336 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .61. Seq ID No.197 is a hypomethylated probe targeting the target region shown in Seq ID No.62, and Seq ID No.337 is a hypermethylated probe targeting the target region shown in Seq ID No.62. Seq ID No. 198 is a hypomethylated probe targeting the target region shown in Seq ID No. 63, and Seq ID No. 338 is a hypermethylated probe targeting the target region shown in Seq ID No. 63. Seq ID No. 199 is a hypomethylated probe targeting the target region shown in Seq ID No. 64, and Seq ID No. 339 is a hypermethylated probe targeting the target region shown in Seq ID No. 64. Seq ID No. 200 is a hypomethylated probe targeting the target region shown in Seq ID No. 65, and Seq ID No. 340 is a hypermethylated probe targeting the target region shown in Seq ID No. 65. Seq ID No. 201 is a hypomethylated probe targeting the target region shown in Seq ID No. 66, and Seq ID No. 341 is a hypermethylated probe targeting the target region shown in Seq ID No. 66. Seq ID No. 202 is a hypomethylated probe targeting the target region shown in Seq ID No. 67, and Seq ID No. 342 is a hypermethylated probe targeting the target region shown in Seq ID No. 67. Seq ID No. 203 is a hypomethylated probe targeting the target region shown in Seq ID No. 68, and Seq ID No. 343 is a hypermethylated probe targeting the target region shown in Seq ID No. 68. Seq ID No. 204 is a hypomethylated probe that targets the target region shown in Seq ID No. 69, and Seq ID No. 344 is a hypermethylated probe that targets the target region shown in Seq ID No. 69. Seq ID No. 205 is a hypomethylated probe targeting the target region shown in Seq ID No. 70, and Seq ID No. 345 is a hypermethylated probe targeting the target region shown in Seq ID No. 70. Seq ID No. 206 is a hypomethylated probe targeting the target region shown in Seq ID No. 71, and Seq ID No. 346 is a hypermethylated probe targeting the target region shown in Seq ID No. 71. Seq ID No. 207 is a hypomethylated probe targeting the target region shown in Seq ID No. 72, and Seq ID No. 347 is a hypermethylated probe targeting the target region shown in Seq ID No. 72. Seq ID No. 208 and Seq ID No. 209 are hypomethylated probes that target the target region shown in Seq ID No. 73, and Seq ID No. 348 and Seq ID No. 349 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .73. Seq ID No. 210 is a hypomethylated probe targeting the target region shown in Seq ID No. 74, and Seq ID No. 350 is a hypermethylated probe targeting the target region shown in Seq ID No. 74. Seq ID No. 211 is a hypomethylated probe targeting the target region shown in Seq ID No. 75, and Seq ID No. 351 is a hypermethylated probe targeting the target region shown in Seq ID No. 75. Seq ID No. 212 is a hypomethylated probe targeting the target region shown in Seq ID No. 76, and Seq ID No. 352 is a hypermethylated probe targeting the target region shown in Seq ID No. 76. Seq ID No. 213 is a hypomethylated probe targeting the target region shown in Seq ID No. 77, and Seq ID No. 353 is a hypermethylated probe targeting the target region shown in Seq ID No. 77. Seq ID No. 214 is a hypomethylated probe that targets the target region shown in Seq ID No. 78, and Seq ID No. 354 is a hypermethylated probe that targets the target region shown in Seq ID No. 78. Seq ID No. 215 is a hypomethylated probe targeting the target region shown in Seq ID No. 79, and Seq ID No. 355 is a hypermethylated probe targeting the target region shown in Seq ID No. 79. Seq ID No.216 is a hypomethylated probe targeting the target region shown in Seq ID No.80, and Seq ID No.356 is a hypermethylated probe targeting the target region shown in Seq ID No.80. Seq ID No. 217 is a hypomethylated probe targeting the target region shown in Seq ID No. 81, and Seq ID No. 357 is a hypermethylated probe targeting the target region shown in Seq ID No. 81. Seq ID No. 218 is a hypomethylated probe targeting the target region shown in Seq ID No. 82, and Seq ID No. 358 is a hypermethylated probe targeting the target region shown in Seq ID No. 82. Seq ID No. 219 is a hypomethylated probe targeting the target region shown in Seq ID No. 83, and Seq ID No. 359 is a hypermethylated probe targeting the target region shown in Seq ID No. 83. Seq ID No. 220 is a hypomethylated probe targeting the target region shown in Seq ID No. 84, and Seq ID No. 360 is a hypermethylated probe targeting the target region shown in Seq ID No. 84. Seq ID No. 221 is a hypomethylated probe targeting the target region shown in Seq ID No. 85, and Seq ID No. 361 is a hypermethylated probe targeting the target region shown in Seq ID No. 85. Seq ID No. 222 is a hypomethylated probe targeting the target region shown in Seq ID No. 86, and Seq ID No. 362 is a hypermethylated probe targeting the target region shown in Seq ID No. 86. Seq ID No. 223 is a hypomethylated probe that targets the target region shown in Seq ID No. 87, and Seq ID No. 363 is a hypermethylated probe that targets the target region shown in Seq ID No. 87. Seq ID No. 224 is a hypomethylated probe targeting the target region shown in Seq ID No. 88, and Seq ID No. 364 is a hypermethylated probe targeting the target region shown in Seq ID No. 88. Seq ID No. 225 and Seq ID No. 226 are both hypomethylated probes that target the target region shown in Seq ID No. 89, and Seq ID No. 365 and Seq ID No. 366 are both targeted to Seq ID No. The hypermethylated probe in the target region shown in .89. Seq ID No. 227 is a hypomethylated probe that targets the target region shown in Seq ID No. 90, and Seq ID No. 367 is a hypermethylated probe that targets the target region shown in Seq ID No. 90. Seq ID No. 228 is a hypomethylated probe that targets the target region shown in Seq ID No. 91, and Seq ID No. 368 is a hypermethylated probe that targets the target region shown in Seq ID No. 91. Seq ID No. 229 is a hypomethylated probe targeting the target region shown in Seq ID No. 92, and Seq ID No. 369 is a hypermethylated probe targeting the target region shown in Seq ID No. 92. Seq ID No. 230, Seq ID No. 231, and Seq ID No. 232 are all hypomethylated probes that target the target region shown in Seq ID No. 93, Seq ID No. 370, Seq ID No. 371 and Seq ID No. 371. Seq ID No. 372 are all hypermethylated probes that target the target region shown in Seq ID No. 93. Seq ID No. 233 is a hypomethylated probe targeting the target region shown in Seq ID No. 94, and Seq ID No. 373 is a hypermethylated probe targeting the target region shown in Seq ID No. 94. Seq ID No. 234 is a hypomethylated probe targeting the target region shown in Seq ID No. 95, and Seq ID No. 374 is a hypermethylated probe targeting the target region shown in Seq ID No. 95. Seq ID No. 235 is a hypomethylated probe targeting the target region shown in Seq ID No. 96, and Seq ID No. 375 is a hypermethylated probe targeting the target region shown in Seq ID No. 96. Seq ID No. 236 and Seq ID No. 237 are hypomethylated probes that target the target region shown in Seq ID No. 97, and Seq ID No. 376 and Seq ID No. 377 are both targeted to Seq ID No. A hypermethylated probe in the target region shown in .97. Seq ID No. 238 is a hypomethylated probe targeting the target region shown in Seq ID No. 98, and Seq ID No. 378 is a hypermethylated probe targeting the target region shown in Seq ID No. 98. Seq ID No. 239 is a hypomethylated probe targeting the target region shown in Seq ID No. 99, and Seq ID No. 379 is a hypermethylated probe targeting the target region shown in Seq ID No. 99. Seq ID No. 240 is a hypomethylated probe targeting the target region shown in Seq ID No. 100, and Seq ID No. 380 is a hypermethylated probe targeting the target region shown in Seq ID No. 100. Table 1 shows the target sequence targeted by the probe.
在本文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 herein, 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.
本文还提供了一种试剂盒。This article also provides a kit.
在本文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 herein, the kit includes the probe composition according to any one of the above embodiments.
本文还提供了探针组合物的用途。The use of the probe composition is also provided herein.
在本文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、肝癌和胰腺癌的试剂盒。In a specific embodiment herein, the above-mentioned probe composition is used to prepare a kit for detecting esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer.
本文还提供了一种芯片。This article also provides a chip.
在本文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 herein, the probe composition according to any one of the above embodiments is immobilized on the chip.
本文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、肝癌和胰腺癌等6种癌症的方法。This article also provides a method for detecting six cancers, including esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic cancer, by using the probe composition.
在本文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6种恶性肿瘤(食管癌、胃癌、结直肠癌、肺癌、肝癌和胰腺癌)。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 herein, the detection method uses hybrid capture to enrich cfDNA, and uses NGS technology to detect methylation sites that are highly related to cancer. Covering the six malignant tumors with the highest incidence in my country (esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer and pancreatic 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6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 above six cancers by using the kit.
具体来说，本申请涉及受试者6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 six 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.：65-100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.：101-197中的任意，靶向泛癌特异性区域的探针Seq ID No.：198-199中的任意，和靶向组织特异性区域的探针Seq ID No.：200-240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.：241-337中的任意，靶向泛癌特异性区域的探针Seq ID No.：338-339中的任意，和靶向组织特异性区域的探针Seq ID No.：340-380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 65-100. The hypomethylation probes include probes targeting cancer-specific regions Seq ID No.: any of 101-197, and probes targeting pan-cancer specific regions Seq ID No.: 198-199 Any, and Seq ID No. of probes targeting tissue-specific regions: Any of 200-240. The hypermethylation probes include probes targeting cancer-specific regions, Seq ID No.: 241-337, and probes targeting pan-cancer specific regions, Seq ID No.: 338-339, Seq ID No.: any of 340-380 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 PCTCN2021081275-appb-000001
Figure PCTCN2021081275-appb-000001
Figure PCTCN2021081275-appb-000002
Figure PCTCN2021081275-appb-000002
Figure PCTCN2021081275-appb-000003
Figure PCTCN2021081275-appb-000003
Figure PCTCN2021081275-appb-000004
Figure PCTCN2021081275-appb-000004
Figure PCTCN2021081275-appb-000005
Figure PCTCN2021081275-appb-000005
Figure PCTCN2021081275-appb-000006
Figure PCTCN2021081275-appb-000006
Figure PCTCN2021081275-appb-000007
Figure PCTCN2021081275-appb-000007
Figure PCTCN2021081275-appb-000008
Figure PCTCN2021081275-appb-000008
Figure PCTCN2021081275-appb-000009
Figure PCTCN2021081275-appb-000009
Figure PCTCN2021081275-appb-000010
Figure PCTCN2021081275-appb-000010
Figure PCTCN2021081275-appb-000011
Figure PCTCN2021081275-appb-000011
Figure PCTCN2021081275-appb-000012
Figure PCTCN2021081275-appb-000012
Figure PCTCN2021081275-appb-000013
Figure PCTCN2021081275-appb-000013
Figure PCTCN2021081275-appb-000014
Figure PCTCN2021081275-appb-000014
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 PCTCN2021081275-appb-000015
Figure PCTCN2021081275-appb-000015
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 PCTCN2021081275-appb-000016
Figure PCTCN2021081275-appb-000016
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 PCTCN2021081275-appb-000017
Figure PCTCN2021081275-appb-000017
Figure PCTCN2021081275-appb-000018
Figure PCTCN2021081275-appb-000018
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, and place the PCR tube on the magnetic stand for 3 minutes to clarify 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200μl 80％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 200μl of 80% ethanol solution to the PCR tube, and let it stand for 30s.
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 PCTCN2021081275-appb-000019
Figure PCTCN2021081275-appb-000019
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 PCTCN2021081275-appb-000020
Figure PCTCN2021081275-appb-000020
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 PCTCN2021081275-appb-000021
Figure PCTCN2021081275-appb-000021
Figure PCTCN2021081275-appb-000022
Figure PCTCN2021081275-appb-000022
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.：101-197中的任意，b)靶向泛癌特异性区域的探针Seq ID No.：198-199中的任意，和c)靶向组织特异性区域的探针Seq ID No.：200-240中的任意，a) Seq ID No. of probes targeting cancer-specific regions: any of 101-197, b) Seq ID No. of probes targeting cancer-specific regions: any of 198-199, and c) Seq ID No. of probes targeting tissue-specific regions: Any of 200-240,
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.：241-337中的任意，e)靶向泛癌特异性区域的探针Seq ID No.：338-339中的任意，和f)靶向组织特异性区域的探针Seq ID No.：340-380中的任意。d) Seq ID No. of probes targeting cancer-specific regions: any of 241-337, e) Seq ID No. of probes targeting cancer-specific regions: any of 338-339, and f) Seq ID No. of probes targeting tissue-specific regions: any of 340-380.
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 PCTCN2021081275-appb-000023
Figure PCTCN2021081275-appb-000023
Figure PCTCN2021081275-appb-000024
Figure PCTCN2021081275-appb-000024
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 PCTCN2021081275-appb-000025
Figure PCTCN2021081275-appb-000025
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.75Seq ID No.75
CPECPE 44 166300242166300242 166300291166300291 0.420.42 Seq ID No.76Seq ID No. 76
PLXDC2PLXDC2 1010 2010449720104497 2010454620104546 0.460.46 Seq ID No.84Seq ID No. 84
PLXDC2PLXDC2 1010 2010475820104758 2010480720104807 0.460.46 Seq ID No.85Seq ID No. 85
PLXDC2PLXDC2 1010 2010494820104948 2010499720104997 0.530.53 Seq ID No.86Seq ID No. 86
PLXDC2PLXDC2 1010 2010559320105593 2010564220105642 0.490.49 Seq ID No.87Seq ID No.87
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.75、Seq ID No.76、Seq ID No.84、Seq ID No.85、Seq ID No.86和Seq ID No.87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. 75, Seq ID No. 76, Seq ID No. 84, Seq ID No. 85, Seq ID No. 86 and Seq ID No. 87 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.78Seq ID No. 78
C6orf155C6orf155 66 7213055372130553 7213060272130602 0.710.71 Seq ID No.79Seq ID No. 79
C6orf155C6orf155 66 7213064172130641 7213069072130690 0.650.65 Seq ID No.80Seq ID No. 80
C6orf155C6orf155 66 7213075572130755 7213080472130804 0.690.69 Seq ID No.81Seq ID No.81
SHISA2SHISA2 1313 2662527326625273 2662539726625397 0.610.61 Seq ID No.93Seq ID No.93
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.78、Seq ID No.79、Seq ID No.80、Seq ID No.81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. 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 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 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. 78, Seq ID No. 79 , Seq ID No. 80, Seq ID No. 81 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.69Seq ID No. 69
FMO3FMO3 11 171060010171060010 171060059171060059 0.330.33 Seq ID No.70Seq ID No. 70
ITIH3ITIH3 33 5282874652828746 5282881952828819 0.250.25 Seq ID No.73Seq ID No. 73
CHST12CHST12 77 24735292473529 24735782473578 0.510.51 Seq ID No.82Seq ID No. 82
CHST12CHST12 77 24738112473811 24738602473860 0.510.51 Seq ID No.83Seq ID No. 83
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.69、Seq ID No.70、Seq ID No.73、Seq ID No.82和Seq ID No.83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. 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 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, 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 21, the target regions that are greater than or equal to their respective thresholds, it can be seen that there are 5 target regions specific to lung tissue: Seq ID No. 69, Seq ID No. 70, Seq ID No. 73, Seq ID No. 82, and Seq ID No. 83 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, The lung tissue-specific markers are the most, and 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 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 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.71Seq ID No. 71
GLI2GLI2 22 121570226121570226 121570275121570275 0.430.43 Seq ID No.72Seq ID No. 72
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 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.71和Seq ID No.72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. 71 and Seq ID No. 72 that are specific to liver tissue are greater than or equal to the respective thresholds of the methylation level, and there is no other tissue 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 PCTCN2021081275-appb-000026
Figure PCTCN2021081275-appb-000026
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.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，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 belong to the esophageal tissue-specific Seq ID No. 66, Seq ID No. 67, and Seq ID No. 74 are greater than or equal to their respective methylation levels. Threshold, 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, the esophageal tissue-specific markers are the most, and the final judgment is The sample is 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.94Seq ID No.94
FRYFRY 1313 3260556332605563 3260561232605612 0.400.40 Seq ID No.95Seq ID No. 95
FRYFRY 1313 3260590232605902 3260595132605951 0.390.39 Seq ID No.96Seq ID No. 96
BRF1BRF1 1414 105714785105714785 105714844105714844 0.330.33 Seq ID No.97Seq ID No. 97
BRF1BRF1 1414 105715025105715025 105715074105715074 0.380.38 Seq ID No.98Seq ID No. 98
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.94、Seq ID No.95、Seq ID No.96、Seq ID No.97和Seq ID No.98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. 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 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, 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 24, the target regions that are greater than or equal to their respective thresholds, it can be seen that there are 5 target regions Seq ID No. 94, Seq ID No. 95, Seq ID No. 96, Seq ID No. 97, and Seq ID that are specific to pancreatic tissue No. 98 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, 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.
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 (17)

  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、肝癌和胰腺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, gastric cancer, colorectal cancer, lung cancer, liver cancer, and pancreatic 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的探针，Probes targeting specific regions of pan-cancer,
    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的探针，Probes that target specific regions of the tissue,
    所述组织特异性区域选自Seq ID No.：65-100中的任意。The tissue-specific region is selected from any of Seq ID No.: 65-100.
  4. 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.：66-67、74-76、84-89、99-100是食管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.: 66-67, 74-76, 84-89, 99-100 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，在所述组织特异性区域中，Seq ID No.：75、76、84-89是胃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.: 75, 76, 84-89 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，在所述组织特异性区域中，Seq ID No.：77-81、93是结直肠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.: 77-81, 93 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，在所述组织特异性区域中，Seq ID No.：65、68-70、73、82-83、90-91是肺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.: 65, 68-70, 73, 82-83, 90-91 are lung tissue-specific Sexual target area.
  8. 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.：71-72、92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.: 71-72, 92 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，在所述组织特异性区域中，Seq ID No.：94-98是胰腺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.: 94-98 is a tissue-specific target region of the pancreas.
  10. 根据权利要求1-9中任一项所述的探针组合物，其包括：The probe composition according to any one of claims 1-9, 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A hypomethylation probe that hybridizes to the cancer-specific region, pan-cancer-specific region, and tissue-specific region that are converted by bisulfite without 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。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.
  11. 根据权利要求10所述的探针组合物，其中所述探针组合物中的每一个探针的长度为40～60bp。The probe composition according to claim 10, wherein the length of each probe in the probe composition is 40-60 bp.
  12. 根据权利要求11所述的探针组合物，其中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。The probe composition according to claim 11, wherein the length of each probe in the probe composition is 45-56 bp, preferably 50-56 bp, and more preferably 50 bp.
  13. 根据权利要求10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.：101-197中的任意，靶向泛癌特异性区域的探针Seq ID No.：198-199中的任意，和靶向组织特异性区域的探针Seq ID No.：200-240中的任意。The probe composition according to claim 10, wherein the hypomethylation probe comprises a probe targeting a cancer-specific region. Seq ID No.: any of 101-197, targeting pan-cancer Seq ID No. of probes for specific regions: any of 198-199, and Seq ID No. of probes for tissue-specific regions: any of 200-240.
  14. 根据权利要求10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.：241-337中的任意，靶向泛癌特异性区域的探针Seq ID No.：338-339中的任意，和靶向组织特异性区域的探针Seq ID No.：340-380中的任意。The probe composition according to claim 10, wherein the hypermethylation probe comprises a probe targeting a cancer-specific region Seq ID No.: any of 241-337, targeting pan-cancer specificity The probe Seq ID No. of the region: any of 338-339, and the probe Seq ID No. of the tissue-specific region: any of 340-380.
  15. 一种试剂盒，其包括权利要求1-14中任一项所述的探针组合物。A kit comprising the probe composition according to any one of claims 1-14.
  16. 一种权利要求1-14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、肝癌和胰腺癌的试剂盒中的应用。An application of the probe composition of any one of claims 1-14 in the preparation of a kit for detecting esophageal cancer, gastric cancer, colorectal cancer, lung cancer, liver cancer and pancreatic cancer.
  17. 一种芯片，其上固定有包括权利要求1-14中任一项所述的探针组合物。A chip on which the probe composition according to any one of claims 1-14 is immobilized.
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