CN117448450A

CN117448450A - Marker for colorectal cancer screening, probe composition and application thereof

Info

Publication number: CN117448450A
Application number: CN202210879055.9A
Authority: CN
Inventors: 李永君; 魏闯; 王秀秀; 吴宁宁; 杨亚东; 郭媛媛; 刘栓平; 王翠翠; 周光朋; 吴振
Original assignee: Biochain Beijing Science and Technology Inc
Current assignee: Biochain Beijing Science and Technology Inc
Priority date: 2022-07-25
Filing date: 2022-07-25
Publication date: 2024-01-26

Abstract

The application discloses a marker for colorectal cancer screening, a probe composition and application thereof, wherein the marker is selected from any one of 44 markers. The marker can sensitively and specifically detect the methylation state of the gene, so that the marker can be used for detecting free DNA of peripheral blood, and the composition can be used for screening asymptomatic people in a non-invasive mode, reduces the harm caused by invasive detection, has higher sensitivity and accuracy and can realize real-time monitoring.

Description

Marker for colorectal cancer screening, probe composition and application thereof

Technical Field

The application relates to the field of biotechnology, in particular to a marker for colorectal cancer screening, a probe composition and application thereof.

Background

Colorectal cancer (colorectal cancer, CRC) is a common malignant tumor, the incidence and mortality rate are both rising, the number of 40.1 ten thousand diagnosed cases in 2016 is increased to 45.6 ten thousand diagnosed cases in 2020, and the composite annual growth rate is 3.3%. It is expected that the number of colorectal cancer episodes will reach 48.4 thousand diagnosed cases in 2022. In 2020, the incidence of colorectal cancer is ranked second among all cancers in China, and the number of deaths of colorectal cancer in China in the same year reaches 28.6 ten thousand. Colorectal cancer is classified into stages I-IV according to the stage of disease progression. For patients with stage IV colorectal cancer, overall survival is very low, with survival rates of less than 10% for 5 years. And patients in stage I can reach more than 90% survival rate. Therefore, early diagnosis and treatment are important in order to improve the overall prognosis of intestinal cancer. At present, the Chinese colorectal cancer screening technology mainly comprises fecal occult blood test, tumor marker detection and enteroscopy. Colonoscopes are still the fundamental method of confirming bowel cancer, but their high cost, poor compliance and potential trauma make them difficult to apply on a large scale for screening of asymptomatic people. Fecal Occult Blood Test (FOBT) and peripheral blood carcinoembryonic antigen (CEA), although acceptable, their poor sensitivity and specificity also present challenges for early diagnosis and treatment of intestinal cancer.

Disclosure of Invention

The object of the present application is to provide a marker for detecting colorectal cancer, which can be used for screening colorectal cancer, is used for screening asymptomatic population in a non-invasive way, is used for prognosis detection of cancer patients, reduces the harm caused by invasive detection, and has higher sensitivity and accuracy, and a probe composition.

The specific technical scheme of the application is as follows:

1. a marker for detecting colorectal cancer, characterized in that the gene to which the marker corresponds is selected from one of the following: ANKRD20A12P, C orf127, HSPA7, ADRA2A, MTRNR L8, MUS81, RIMBP2, WNK1, EDDM3A, TBPL2, ARNT2, MIR1282, FZD2, LOC339166, ANKRD62, ISOC2, LENG9, ABCG8, CDRT15P3, LAPTM4A, ADAMTS5, MIR3648, RFPL2, ANKRD18DP, FAM86HP, DUX4L8, KIAA0825, PCBD2, PCDHB3, PCDHGA1, PSORS1C2, LINC02902, LUARIS, ATAD2, NAT1, NA1B and UCK1.

2. The marker according to item 1, wherein the nucleotide sequence of the marker is selected from one of the markers shown in SEQ ID NO. 1-44, preferably the marker is a methylated marker.

3. A probe composition comprising a probe that targets methylation of a marker of item 1 or 2.

4. The probe composition of item 3, wherein the probe composition comprises a hypermethylated first probe composition for hybridization to a bisulfite converted hypermethylated region and a hypomethylated second probe composition for hybridization to a bisulfite converted hypomethylated region;

preferably, the first probe composition comprises n probes that hybridize to each nucleotide of the sense strand and/or the antisense strand of the bisulfite converted hypermethylated region;

preferably, the second probe composition comprises m probes that hybridize to each nucleotide of the sense strand and/or the antisense strand of the bisulfite converted hypomethylated region;

preferably, n and m are each any integer from 1 to 10;

preferably, there is x between the n-1 th probe and the n-th probe ₁ Individual coresNucleotide overlap, preferably x ₁ Is any integer from 0 to 100;

preferably, there is x between the m-1 th probe and the m-th probe ₂ Overlapping of nucleotides, preferably x ₂ Is any integer from 0 to 100;

further preferably, the first probe composition comprises one or two of SEQ ID NOS: 45-132 and the second probe composition comprises one or two of SEQ ID NOS: 133-220.

5. Use of a marker for the preparation of a kit for detecting colorectal cancer, characterized in that the gene to which the marker corresponds is selected from one of the following: ANKRD20A12P, C orf127, HSPA7, ADRA2A, MTRNR L8, MUS81, RIMBP2, WNK1, EDDM3A, TBPL2, ARNT2, MIR1282, FZD2, LOC339166, ANKRD62, ISOC2, LENG9, ABCG8, CDRT15P3, LAPTM4A, ADAMTS5, MIR3648, RFPL2, ANKRD18DP, FAM86HP, DUX4L8, KIAA0825, PCBD2, PCDHB3, PCDHGA1, PSORS1C2, LINC02902, LUARIS, ATAD2, NAT1, NA1B and UCK1.

6. The use according to item 5, characterized in that the nucleotide sequence of the marker is selected from one of the markers shown in SEQ ID NO. 1-44, preferably the marker is a methylated marker;

preferably, the probe composition is for targeting a post-methylation marker of colorectal cancer;

preferably, the probe composition is the probe composition of item 3 or 4.

7. A composition for colorectal cancer detection, comprising a nucleic acid for detecting any methylation of a gene corresponding to a marker selected from the group consisting of: ANKRD20A12P, C orf127, HSPA7, ADRA2A, MTRNR L8, MUS81, RIMBP2, WNK1, EDDM3A, TBPL2, ARNT2, MIR1282, FZD2, LOC339166, ANKRD62, ISOC2, LENG9, ABCG8, CDRT15P3, LAPTM4A, ADAMTS5, MIR3648, RFPL2, ANKRD18DP, FAM86HP, DUX4L8, KIAA0825, PCBD2, PCDHB3, PCDHGA1, PSORS1C2, LINC02902, LUARIS, ATAD2, NAT1, NA1B and UCK1.

8. The composition of item 7, wherein the nucleotide sequence of the marker is selected from one of the nucleotide sequences set forth in SEQ ID NOS.1-44.

9. The composition of item 7 or 8, wherein the nucleic acid comprises the probe composition of item 3 or 4;

preferably, the nucleic acid comprises:

a primer that is a fragment of at least 9 nucleotides in a target sequence of the marker, the fragment comprising at least one CpG dinucleotide sequence;

preferably, the nucleic acid further comprises:

a probe that hybridizes under moderately stringent or stringent conditions to at least 15 nucleotide fragments in a target sequence of the marker, the fragments comprising at least one CpG dinucleotide sequence;

Preferably, the composition further comprises an agent that converts the unmethylated cytosine base at position 5 of the target sequence of the marker to uracil;

preferably, the nucleic acid for detecting methylation of a target sequence of a marker further comprises:

blocking agents that preferentially bind to target sequences in the unmethylated state.

10. A kit comprising a reagent for detecting a marker according to item 1 or 2 or a probe composition according to item 3 or 4 or a composition according to any one of items 7 to 9.

11. A chip comprising the marker of item 1 or 2 or the probe composition of item 3 or 4 or the composition of item 7 or 8.

ADVANTAGEOUS EFFECTS OF INVENTION

The inventor of the application utilizes epigenetic and bioinformatics technology to find a plurality of methylation genes related to colorectal cancer by analyzing genome methylation data of the colorectal cancer, determines a target sequence of methylation abnormality of the methylation genes of the colorectal cancer, and can sensitively and specifically detect the methylation state of the genes through the target sequence of the methylation genes, so that the methylation genes can be used for detecting free DNA of peripheral blood.

The composition is used for screening asymptomatic people in a non-invasive mode, harm caused by invasive detection is reduced, and the composition has higher sensitivity and accuracy and can realize real-time monitoring.

Detailed Description

The present application is described in detail below. While specific embodiments of the present application are shown, it should be understood that the present application may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

It should be noted that, throughout the specification and claims, the terms "include" and "comprising" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description hereinafter sets forth the preferred embodiment for carrying out the present application, but is not intended to limit the scope of the present application in general, as the description proceeds. The scope of the present application is defined by the appended claims.

The application provides a marker for detecting colorectal cancer, wherein a gene corresponding to the marker is selected from one of the following: ANKRD20A12P, C orf127, HSPA7, ADRA2A, MTRNR L8, MUS81, RIMBP2, WNK1, EDDM3A, TBPL2, ARNT2, MIR1282, FZD2, LOC339166, ANKRD62, ISOC2, LENG9, ABCG8, CDRT15P3, LAPTM4A, ADAMTS5, MIR3648, RFPL2, ANKRD18DP, FAM86HP, DUX4L8, KIAA0825, PCBD2, PCDHB3, PCDHGA1, PSORS1C2, LINC02902, LUARIS, ATAD2, NAT1, NA1B and UCK1. The corresponding gene refers to a gene corresponding to the marker.

In one embodiment, the nucleotide sequence of the marker is selected from one of the markers shown in SEQ ID NOS.1-44, preferably the marker is a methylated marker.

Wherein the nucleotide sequence of ANKRD20A12P is shown as SEQ ID NO. 1; the nucleotide sequence of the C1orf127 is shown as SEQ ID NO. 2; the nucleotide sequence of the HSPA7 is shown as SEQ ID NO. 3; the nucleotide sequence of ADRA2A is shown as SEQ ID NO. 4; the nucleotide sequence of MTRNR2L8 is shown as SEQ ID NO. 5; the nucleotide sequence of MUS81 is shown as SEQ ID NO. 6; the nucleotide sequence of RIMBP2 is shown as SEQ ID NO. 7; the nucleotide sequence of WNK1 is shown as SEQ ID NO. 8; the nucleotide sequence of EDDM3A is shown in SEQ ID NO 9; the nucleotide sequence of TBPL2 is shown as SEQ ID NO. 10; the nucleotide sequence of ARNT2 is shown in SEQ ID NO. 11; the nucleotide sequence of MIR1282 is shown as SEQ ID NO. 12; the nucleotide sequence of FZD2 is shown as SEQ ID NO. 13; the nucleotide sequence of LOC339166 is shown in SEQ ID NO. 14; the nucleotide sequence of ANKRD62 is shown as SEQ ID NO. 15; the nucleotide sequence of the ISOC2 is shown as SEQ ID NO. 16; the nucleotide sequence of LENG9 is shown as SEQ ID NO. 17; the nucleotide sequence of ABCG8 is shown as SEQ ID NO. 18; the nucleotide sequences of ANKRD18DP are respectively shown as SEQ ID NO. 19 and SEQ ID NO. 20; the nucleotide sequence of LAPTM4A is shown as SEQ ID NO. 21; the nucleotide sequence of ADAMTS5 is shown as SEQ ID NO. 22; the nucleotide sequence of MIR3648 is shown as SEQ ID NO. 23; the nucleotide sequences of RFPL2 are shown as SEQ ID NO. 24 and SEQ ID NO. 25 respectively; the nucleotide sequences of ANKRD18DP are respectively shown as SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28 and SEQ ID NO. 29; the nucleotide sequence of FAM86HP is shown as SEQ ID NO. 30; the nucleotide sequence of DUX4L8 is shown as SEQ ID NO. 31; the nucleotide sequence of KIAA0825 is shown as SEQ ID NO. 32; the nucleotide sequence of PCBD2 is shown as SEQ ID NO. 33; the nucleotide sequence of PCDHB3 is shown as SEQ ID NO. 34; the nucleotide sequences of PCDHGA1 are respectively shown as SEQ ID NO. 35 and SEQ ID NO. 36; the nucleotide sequence of PSORS1C2 is shown as SEQ ID NO. 37; the nucleotide sequence of LINC02902 is shown as SEQ ID NO. 38; the nucleotide sequence of LUARIS is shown as SEQ ID NO. 39; the nucleotide sequence of ATAD2 is shown as SEQ ID NO. 40; the nucleotide sequence of NAT1 is shown as SEQ ID NO. 41; the nucleotide sequences of CACNA1B are shown as SEQ ID NO. 42 and SEQ ID NO. 43 respectively; the nucleotide sequence of UCK1 is shown as SEQ ID NO. 44.

Wherein the sequence of the marker is a sequence which is not converted by bisulfite.

The present application provides a probe composition comprising a probe that targets methylation of the marker.

Methylation refers to methylation of the 5 th carbon atom on cytosine in CpG dinucleotides, and is taken as a stable modification state, and can inherit new generation progeny DNA along with the replication process of DNA under the action of DNA methyltransferase, so that the methylation of the gene promoter region can lead to silence transcription of cancer suppressor genes during DNA methylation, and the methylation is closely related to tumor occurrence. Aberrant methylation includes hypermethylation of cancer suppressor genes and DNA repair genes, hypomethylation of repeated sequence DNA, imprinting loss of certain genes, which are associated with the occurrence of a variety of tumors.

Methylation as described herein can be methylation level, degree of methylation, or methylation state, and when analyzing methylation of such target sequences, one skilled in the art can use quantitative determination methods to determine methylation.

The probe is single-stranded or double-stranded DNA with a length of tens to hundreds or even thousands of base pairs, which can utilize the denaturation, renaturation and high precision of base complementary pairing of molecules, and can be combined with (hybridized with) complementary unlabeled single-stranded DNA or RNA in a sample to be tested in a hydrogen bond manner to form a double-stranded complex (hybrid). After washing off the unpaired and bound probe, the hybridization reaction results can be detected by a detection system such as an autoradiography or an enzyme-linked reaction. In this application, the region that complementarily binds or hybridizes to a probe is a specific target region, and a plurality of probes are combined into a probe composition.

In one embodiment, the probe composition comprises a hypermethylated first probe composition for hybridization to a bisulfite converted hypermethylated region and a hypomethylated second probe composition for hybridization to a bisulfite converted hypomethylated region.

The hypermethylation means that after the marker is converted by bisulfite, a base C is changed into a base U, but if the marker is a base CG, the base C is kept unchanged;

the hypomethylation means that after the marker is converted by bisulfite, all bases CG are not methylated, and the bases C are changed into the bases U.

Since the methylation status varies from person to person, the sequence of the tag converted by bisulfite varies, one extreme case of each tag is shown here, i.e. all CG of the segment is in hypermethylation status, and the hypermethylation status sequence of its complementary strand:

the sequence of one extreme case of SEQ ID NO. 1 is shown as SEQ ID NO. 221;

the sequence of one extreme of the complementary strand of SEQ ID NO. 1 is shown as SEQ ID NO. 222;

the sequence of one extreme case of SEQ ID NO. 2 is shown as SEQ ID NO. 223;

The sequence of one extreme of the complementary strand of SEQ ID NO. 2 is shown as SEQ ID NO. 224;

the sequence of one extreme case of SEQ ID NO. 3 is shown as SEQ ID NO. 225;

the sequence of one extreme of the complementary strand of SEQ ID NO. 3 is shown as SEQ ID NO. 226;

the sequence of one extreme case of SEQ ID NO. 4 is shown as SEQ ID NO. 227;

the sequence of one extreme of the complementary strand of SEQ ID NO. 4 is shown as SEQ ID NO. 228;

the sequence of one extreme case of SEQ ID NO. 5 is shown as SEQ ID NO. 229;

the sequence of one extreme of the complementary strand of SEQ ID NO. 5 is shown as SEQ ID NO. 230;

the sequence of one extreme case of SEQ ID NO. 6 is shown as SEQ ID NO. 231;

the sequence of one extreme of the complementary strand of SEQ ID NO. 6 is shown as SEQ ID NO. 232;

the sequence of one extreme case of SEQ ID NO. 7 is shown as SEQ ID NO. 233;

the sequence of one extreme of the complementary strand of SEQ ID NO. 7 is shown as SEQ ID NO. 234;

the sequence of one extreme case of SEQ ID NO. 8 is shown as SEQ ID NO. 235;

the sequence of one extreme of the complementary strand of SEQ ID NO. 8 is shown as SEQ ID NO. 236;

the sequence of one extreme case of SEQ ID NO. 9 is shown as SEQ ID NO. 237;

the sequence of one extreme of the complementary strand of SEQ ID NO. 9 is shown as SEQ ID NO. 238;

The sequence of one extreme case of SEQ ID NO. 10 is shown as SEQ ID NO. 239;

the sequence of one extreme of the complementary strand of SEQ ID NO. 10 is shown as SEQ ID NO. 240;

the sequence of one extreme case of SEQ ID NO. 11 is shown as SEQ ID NO. 241;

the sequence of one extreme of the complementary strand of SEQ ID NO. 11 is shown as SEQ ID NO. 242;

the sequence of one extreme case of SEQ ID NO. 12 is shown as SEQ ID NO. 243;

the sequence of one extreme of the complementary strand of SEQ ID NO. 12 is shown as SEQ ID NO. 244;

the sequence of one extreme case of SEQ ID NO. 13 is shown as SEQ ID NO. 245;

the sequence of one extreme of the complementary strand of SEQ ID NO. 13 is shown as SEQ ID NO. 246;

the sequence of one extreme case of SEQ ID NO. 14 is shown as SEQ ID NO. 247;

the sequence of one extreme of the complementary strand of SEQ ID NO. 14 is shown as SEQ ID NO. 248;

the sequence of one extreme case of SEQ ID NO. 15 is shown as SEQ ID NO. 249;

the sequence of one extreme of the complementary strand of SEQ ID NO. 15 is shown as SEQ ID NO. 250;

the sequence of one extreme case of SEQ ID NO. 16 is shown as SEQ ID NO. 251;

the sequence of one extreme of the complementary strand of SEQ ID NO. 16 is shown as SEQ ID NO. 252;

the sequence of one extreme case of SEQ ID NO. 17 is shown as SEQ ID NO. 253;

The sequence of one extreme of the complementary strand of SEQ ID NO. 17 is shown as SEQ ID NO. 254;

the sequence of one extreme case of SEQ ID NO. 18 is shown as SEQ ID NO. 255;

the sequence of one extreme of the complementary strand of SEQ ID NO. 18 is shown as SEQ ID NO. 256;

the sequence of one extreme of SEQ ID NO. 19 is shown as SEQ ID NO. 257;

the sequence of one extreme of the complementary strand of SEQ ID NO. 19 is shown as SEQ ID NO. 258;

the sequence of one extreme of SEQ ID NO. 20 is shown as SEQ ID NO. 259;

the sequence of one extreme of the complementary strand of SEQ ID NO. 20 is shown as SEQ ID NO. 260;

the sequence of one extreme case of SEQ ID NO. 21 is shown as SEQ ID NO. 261;

the sequence of one extreme of the complementary strand of SEQ ID NO. 21 is shown as SEQ ID NO. 262;

the sequence of one extreme case of SEQ ID NO. 22 is shown as SEQ ID NO. 263;

the sequence of one extreme of the complementary strand of SEQ ID NO. 22 is shown as SEQ ID NO. 264;

the sequence of one extreme case of SEQ ID NO. 23 is shown as SEQ ID NO. 265;

the sequence of one extreme of the complementary strand of SEQ ID NO. 23 is shown as SEQ ID NO. 266;

the sequence of one extreme case of SEQ ID NO. 24 is shown as SEQ ID NO. 267;

the sequence of one extreme of the complementary strand of SEQ ID NO. 24 is shown as SEQ ID NO. 268;

The sequence of one extreme case of SEQ ID NO. 25 is shown as SEQ ID NO. 269;

the sequence of one extreme of the complementary strand of SEQ ID NO. 25 is shown as SEQ ID NO. 270;

the sequence of one extreme case of SEQ ID NO. 26 is shown as SEQ ID NO. 271;

the sequence of one extreme of the complementary strand of SEQ ID NO. 26 is shown as SEQ ID NO. 272;

the sequence of one extreme case of SEQ ID NO. 27 is shown as SEQ ID NO. 273;

the sequence of one extreme of the complementary strand of SEQ ID NO. 27 is shown as SEQ ID NO. 274;

the sequence of one extreme case of SEQ ID NO. 28 is shown as SEQ ID NO. 275;

the sequence of one extreme of the complementary strand of SEQ ID NO. 28 is shown as SEQ ID NO. 276;

the sequence of one extreme case of SEQ ID NO. 29 is shown as SEQ ID NO. 277;

the sequence of one extreme of the complementary strand of SEQ ID NO. 29 is shown as SEQ ID NO. 278;

the sequence of one extreme case of SEQ ID NO. 30 is shown as SEQ ID NO. 279;

the sequence of one extreme of the complementary strand of SEQ ID NO. 30 is shown as SEQ ID NO. 280;

the sequence of one extreme case of SEQ ID NO. 31 is shown as SEQ ID NO. 281;

the sequence of one extreme of the complementary strand of SEQ ID NO. 31 is shown as SEQ ID NO. 282;

the sequence of one extreme case of SEQ ID NO. 32 is shown as SEQ ID NO. 283;

The sequence of one extreme of the complementary strand of SEQ ID NO. 32 is shown as SEQ ID NO. 284;

the sequence of one extreme case of SEQ ID NO. 33 is shown as SEQ ID NO. 285;

the sequence of one extreme of the complementary strand of SEQ ID NO. 33 is shown as SEQ ID NO. 286;

the sequence of one extreme of SEQ ID NO. 34 is shown as SEQ ID NO. 287;

the sequence of one extreme of the complementary strand of SEQ ID NO. 34 is shown as SEQ ID NO. 288;

the sequence of one extreme case of SEQ ID NO. 35 is shown as SEQ ID NO. 289;

the sequence of one extreme of the complementary strand of SEQ ID NO. 35 is shown as SEQ ID NO. 290;

the sequence of one extreme case of SEQ ID NO. 36 is shown as SEQ ID NO. 291;

the sequence of one extreme of the complementary strand of SEQ ID NO. 36 is shown as SEQ ID NO. 292;

the sequence of one extreme case of SEQ ID NO. 37 is shown as SEQ ID NO. 293;

the sequence of one extreme of the complementary strand of SEQ ID NO. 37 is shown as SEQ ID NO. 294;

the sequence of one extreme case of SEQ ID NO. 38 is shown as SEQ ID NO. 295;

the sequence of one extreme of the complementary strand of SEQ ID NO. 38 is shown as SEQ ID NO. 296;

the sequence of one extreme of SEQ ID NO. 39 is shown as SEQ ID NO. 297;

the sequence of one extreme of the complementary strand of SEQ ID NO. 39 is shown as SEQ ID NO. 298;

The sequence of one extreme case of SEQ ID NO. 40 is shown as SEQ ID NO. 299;

the sequence of one extreme of the complementary strand of SEQ ID NO. 40 is shown as SEQ ID NO. 300;

the sequence of one extreme case of SEQ ID NO. 41 is shown as SEQ ID NO. 301;

the sequence of one extreme of the complementary strand of SEQ ID NO. 41 is shown as SEQ ID NO. 302;

the sequence of one extreme case of SEQ ID NO. 42 is shown as SEQ ID NO. 303;

the sequence of one extreme of the complementary strand of SEQ ID NO. 42 is shown as SEQ ID NO. 304;

the sequence of one extreme case of SEQ ID NO. 43 is shown as SEQ ID NO. 305;

the sequence of one extreme of the complementary strand of SEQ ID NO. 43 is shown as SEQ ID NO. 306;

the sequence of one extreme of SEQ ID NO. 44 is shown as SEQ ID NO. 307;

the sequence of one extreme of the complementary strand of SEQ ID NO. 44 is shown as SEQ ID NO. 308.

Similarly, since each person has a different methylation state, an extreme case is shown here, in which all CG is in hypomethylated state, and the sequence of hypomethylated states of their complementary strands is also shown:

the sequence of one extreme of SEQ ID NO. 1 is shown as SEQ ID NO. 309;

the sequence of one extreme of the complementary strand of SEQ ID NO. 1 is shown as SEQ ID NO. 310;

The sequence of one extreme case of SEQ ID NO. 2 is shown as SEQ ID NO. 311;

the sequence of one extreme of the complementary strand of SEQ ID NO. 2 is shown as SEQ ID NO. 312;

the sequence of one extreme case of SEQ ID NO. 3 is shown as SEQ ID NO. 313;

the sequence of one extreme of the complementary strand of SEQ ID NO. 3 is shown as SEQ ID NO. 314;

the sequence of one extreme case of SEQ ID NO. 4 is shown as SEQ ID NO. 315;

the sequence of one extreme of the complementary strand of SEQ ID NO. 4 is shown as SEQ ID NO. 316;

the sequence of one extreme case of SEQ ID NO. 5 is shown as SEQ ID NO. 317;

the sequence of one extreme of the complementary strand of SEQ ID NO. 5 is shown as SEQ ID NO. 318;

the sequence of one extreme case of SEQ ID NO. 6 is shown as SEQ ID NO. 319;

the sequence of one extreme of the complementary strand of SEQ ID NO. 6 is shown as SEQ ID NO. 320;

the sequence of one extreme case of SEQ ID NO. 7 is shown as SEQ ID NO. 321;

the sequence of one extreme of the complementary strand of SEQ ID NO. 7 is shown as SEQ ID NO. 322;

the sequence of one extreme case of SEQ ID NO. 8 is shown as SEQ ID NO. 323;

the sequence of one extreme of the complementary strand of SEQ ID NO. 8 is shown as SEQ ID NO. 324;

the sequence of one extreme case of SEQ ID NO. 9 is shown as SEQ ID NO. 325;

The sequence of one extreme of the complementary strand of SEQ ID NO. 9 is shown as SEQ ID NO. 326;

the sequence of one extreme case of SEQ ID NO. 10 is shown as SEQ ID NO. 327;

the sequence of one extreme of the complementary strand of SEQ ID NO. 10 is shown as SEQ ID NO. 328;

the sequence of one extreme case of SEQ ID NO. 11 is shown as SEQ ID NO. 329;

the sequence of one extreme of the complementary strand of SEQ ID NO. 11 is shown as SEQ ID NO. 330;

the sequence of one extreme case of SEQ ID NO. 12 is shown as SEQ ID NO. 331;

the sequence of one extreme of the complementary strand of SEQ ID NO. 12 is shown as SEQ ID NO. 332;

the sequence of one extreme case of SEQ ID NO. 13 is shown as SEQ ID NO. 333;

the sequence of one extreme of the complementary strand of SEQ ID NO. 13 is shown as SEQ ID NO. 334;

the sequence of one extreme case of SEQ ID NO. 14 is shown as SEQ ID NO. 335;

the sequence of one extreme of the complementary strand of SEQ ID NO. 14 is shown as SEQ ID NO. 336;

the sequence of one extreme case of SEQ ID NO. 15 is shown as SEQ ID NO. 337;

the sequence of one extreme of the complementary strand of SEQ ID NO. 15 is shown as SEQ ID NO. 338;

the sequence of one extreme of SEQ ID NO. 16 is shown as SEQ ID NO. 339;

the sequence of one extreme of the complementary strand of SEQ ID NO. 16 is shown as SEQ ID NO. 340;

The sequence of one extreme case of SEQ ID NO. 17 is shown as SEQ ID NO. 341;

the sequence of one extreme of the complementary strand of SEQ ID NO. 17 is shown as SEQ ID NO. 342;

the sequence of one extreme case of SEQ ID NO. 18 is shown as SEQ ID NO. 343;

the sequence of one extreme of the complementary strand of SEQ ID NO. 18 is shown as SEQ ID NO. 344;

the sequence of one extreme case of SEQ ID NO. 19 is shown as SEQ ID NO. 345;

the sequence of one extreme of the complementary strand of SEQ ID NO. 19 is shown as SEQ ID NO. 346;

the sequence of one extreme case of SEQ ID NO. 20 is shown as SEQ ID NO. 347;

the sequence of one extreme of the complementary strand of SEQ ID NO. 20 is shown as SEQ ID NO. 348;

the sequence of one extreme of SEQ ID NO. 21 is shown as SEQ ID NO. 349;

the sequence of one extreme of the complementary strand of SEQ ID NO. 21 is shown as SEQ ID NO. 350;

the sequence of one extreme case of SEQ ID NO. 22 is shown as SEQ ID NO. 351;

the sequence of one extreme of the complementary strand of SEQ ID NO. 22 is shown as SEQ ID NO. 352;

the sequence of one extreme case of SEQ ID NO. 23 is shown as SEQ ID NO. 353;

the sequence of one extreme of the complementary strand of SEQ ID NO. 23 is shown as SEQ ID NO. 354;

the sequence of one extreme case of SEQ ID NO. 24 is shown as SEQ ID NO. 355;

The sequence of one extreme of the complementary strand of SEQ ID NO. 24 is shown as SEQ ID NO. 356;

the sequence of one extreme case of SEQ ID NO. 25 is shown as SEQ ID NO. 357;

the sequence of one extreme of the complementary strand of SEQ ID NO. 25 is shown as SEQ ID NO. 358;

the sequence of one extreme case of SEQ ID NO. 26 is shown as SEQ ID NO. 359;

the sequence of one extreme of the complementary strand of SEQ ID NO. 26 is shown as SEQ ID NO. 360;

the sequence of one extreme case of SEQ ID NO. 27 is shown as SEQ ID NO. 361;

the sequence of one extreme of the complementary strand of SEQ ID NO. 27 is shown as SEQ ID NO. 362;

the sequence of one extreme case of SEQ ID NO. 28 is shown as SEQ ID NO. 363;

the sequence of one extreme of the complementary strand of SEQ ID NO. 28 is shown as SEQ ID NO. 364;

the sequence of one extreme case of SEQ ID NO. 29 is shown as SEQ ID NO. 365;

the sequence of one extreme of the complementary strand of SEQ ID NO. 29 is shown as SEQ ID NO. 366;

the sequence of one extreme case of SEQ ID NO. 30 is shown as SEQ ID NO. 367;

the sequence of one extreme of the complementary strand of SEQ ID NO. 30 is shown as SEQ ID NO. 368;

the sequence of one extreme case of SEQ ID NO. 31 is shown as SEQ ID NO. 369;

the sequence of one extreme of the complementary strand of SEQ ID NO. 31 is shown as SEQ ID NO. 370;

The sequence of one extreme case of SEQ ID NO. 32 is shown as SEQ ID NO. 371;

the sequence of one extreme of the complementary strand of SEQ ID NO. 32 is shown as SEQ ID NO. 372;

the sequence of one extreme case of SEQ ID NO. 33 is shown as SEQ ID NO. 373;

the sequence of one extreme of the complementary strand of SEQ ID NO. 33 is shown as SEQ ID NO. 374;

the sequence of one extreme case of SEQ ID NO. 34 is shown as SEQ ID NO. 375;

the sequence of one extreme of the complementary strand of SEQ ID NO. 34 is shown as SEQ ID NO. 376;

the sequence of one extreme case of SEQ ID NO. 35 is shown as SEQ ID NO. 377;

the sequence of one extreme of the complementary strand of SEQ ID NO. 35 is shown as SEQ ID NO. 378;

the sequence of one extreme case of SEQ ID NO. 36 is shown as SEQ ID NO. 379;

the sequence of one extreme of the complementary strand of SEQ ID NO. 36 is shown as SEQ ID NO. 380;

the sequence of one extreme case of SEQ ID NO. 37 is shown as SEQ ID NO. 381;

the sequence of one extreme of the complementary strand of SEQ ID NO. 37 is shown as SEQ ID NO. 382;

the sequence of one extreme case of SEQ ID NO. 38 is shown as SEQ ID NO. 383;

the sequence of one extreme of the complementary strand of SEQ ID NO. 38 is shown as SEQ ID NO. 384;

the sequence of one extreme of SEQ ID NO. 39 is shown as SEQ ID NO. 385;

The sequence of one extreme of the complementary strand of SEQ ID NO. 39 is shown as SEQ ID NO. 386;

the sequence of one extreme case of SEQ ID NO. 40 is shown as SEQ ID NO. 387;

the sequence of one extreme of the complementary strand of SEQ ID NO. 40 is shown as SEQ ID NO. 388;

the sequence of one extreme case of SEQ ID NO. 41 is shown as SEQ ID NO. 389;

the sequence of one extreme of the complementary strand of SEQ ID NO. 41 is shown as SEQ ID NO. 390;

the sequence of one extreme case of SEQ ID NO. 42 is shown as SEQ ID NO. 391;

the sequence of one extreme of the complementary strand of SEQ ID NO. 42 is shown as SEQ ID NO. 392;

the sequence of one extreme case of SEQ ID NO. 43 is shown as SEQ ID NO. 393;

the sequence of one extreme of the complementary strand of SEQ ID NO. 43 is shown as SEQ ID NO. 394;

the sequence of one extreme of SEQ ID NO. 44 is shown as SEQ ID NO. 395;

the sequence of one extreme of the complementary strand of SEQ ID NO. 44 is shown as SEQ ID NO. 396.

In one embodiment, the first probe composition comprises n probes that hybridize to each nucleotide of the sense strand and/or the antisense strand of the bisulfite converted hypermethylated region.

The second probe composition includes m probes that hybridize to each nucleotide of the sense strand and/or the antisense strand of the bisulfite converted hypomethylated region.

The number of probes in the first probe composition and the second probe composition is not limited in any way, and those skilled in the art can select the number as desired, for example, m and n may be any integer of 1 to 10, and m and n may be the same or different.

For example, m and n may be any integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, preferably m=n=2.

In one embodiment, there is x between the n-1 th probe and the n-th probe ₁ The number of nucleotides overlap, preferably,x ₁ is any integer from 0 to 100;

preferably, there is x between the m-1 th probe and the m-th probe ₂ Overlapping of nucleotides, preferably x ₂ Is any integer from 0 to 100.

Wherein x is ₁ And x ₂ May be the same or different, when x ₁ When 0, it is indicated that the tail of the n-1 th probe is connected with the head of the n-th probe, and similarly, when x ₂ When 0, it is indicated that the tail of the m-1 th probe is connected to the head of the m-th probe.

The probe composition is hybridized with the marker converted by the bisulfite, wherein the high-methylation first probe composition is hybridized with a high-methylation region, and the low-methylation second probe composition is hybridized with a low-methylation region, so that the methylation level of a target sequence can be detected efficiently and accurately, and the probe composition can be used for colorectal cancer screening.

In one embodiment, the hypermethylated first probe composition comprises one or both of SEQ ID NOS: 45-132.

The hypomethylated second probe composition comprises one or two of SEQ ID NOS: 133-220.

Wherein the first probe composition for hybridization with the ANKRD20A12P methylated sequence comprises the nucleotide sequence as set forth in SEQ ID NOS.45-46;

the first probe composition for hybridization with the C1orf127 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS.47-48;

the first probe composition for hybridization with a HSPA7 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOs 49-50;

the first probe composition for hybridization with ADRA2A methylation sequence comprises a nucleotide sequence as shown in SEQ ID NOS.51-52;

a first probe composition for hybridization with an MTRNR2L8 methylated sequence comprises the nucleotide sequence set forth in SEQ ID NOS.53-54;

the first probe composition for hybridization to the MUS81 methylated sequence comprises the nucleotide sequence shown as SEQ ID NOS.55-56;

the first probe composition for hybridization with the RIMBP2 methylation sequence comprises the nucleotide sequence shown as SEQ ID NOS: 57-58;

the first probe composition for hybridization with the WNK1 methylation sequence comprises a nucleotide sequence as shown in SEQ ID NOS 59-60;

The first probe composition for hybridization with EDDM3A methylation sequence comprises a nucleotide sequence as shown in SEQ ID NO. 61-62;

the first probe composition for hybridization with TBPL2 methylated sequences comprises the nucleotide sequence shown as SEQ ID NOS: 63-64;

the first probe composition for hybridization with ARNT2 methylation sequence comprises the nucleotide sequence set forth in SEQ ID NOS.65-66;

a first probe composition for hybridization to MIR1282 methylation sequences comprises a nucleotide sequence as set forth in SEQ ID NOS 67-68;

the first probe composition for hybridization with the FZD2 methylation sequence includes a nucleotide sequence as shown in SEQ ID NO: 69-70;

a first probe composition for hybridization with LOC339166 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS: 71-72;

a first probe composition for hybridization to the ANKRD62 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS: 73-74;

the first probe composition for hybridization with an ISOC2 methylation sequence comprises a nucleotide sequence set forth in SEQ ID NOs 75-76;

the first probe composition for hybridization to a LENG9 methylation sequence comprises the nucleotide sequence shown in SEQ ID NOS: 77-78;

the first probe composition for hybridization with ABCG8 methylation sequence comprises the nucleotide sequence shown as SEQ ID NO. 79-80; the method comprises the steps of carrying out a first treatment on the surface of the

The first probe composition for hybridization with CDRT15P3 methylation sequences comprises the nucleotide sequence shown as SEQ ID NO. 81-82 (which is used for hybridization with SEQ ID NO. 19) and comprises the nucleotide sequence shown as SEQ ID NO. 83-84 (which is used for hybridization with SEQ ID NO. 20);

the first probe composition for hybridization to LAPTM4A methylation sequence comprises the nucleotide sequence shown in SEQ ID NOS.85-86;

a first probe composition for hybridization with an ADAMTS5 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS: 87-88;

the first probe composition for hybridization with the MIR3648 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS.89-90;

a first probe composition for hybridization with RFPL2 methylation sequences comprises the nucleotide sequences shown as SEQ ID NOS: 91-92 (which are for hybridization with SEQ ID NO: 24) and comprises the nucleotide sequences shown as SEQ ID NOS: 93-94 (which are for hybridization with SEQ ID NO: 25);

a first probe composition for hybridization with the ANKRD18DP methylation sequence comprises the nucleotide sequence shown as SEQ ID NO 95-96 (which is used for hybridization with SEQ ID NO 26), the nucleotide sequence shown as SEQ ID NO 97-98 (which is used for hybridization with SEQ ID NO 27), the nucleotide sequence shown as SEQ ID NO 99-100 (which is used for hybridization with SEQ ID NO 28), and the nucleotide sequence shown as SEQ ID NO 101-102 (which is used for hybridization with SEQ ID NO 29);

A first probe composition for hybridization to FAM86HP methylation sequence comprises the nucleotide sequence shown as SEQ ID NOS.103-104;

a first probe composition for hybridization to a DUX4L8 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS.105-106;

a first probe composition for hybridization with a KIAA0825 methylation sequence comprises the nucleotide sequence shown in SEQ ID NOS.107-108;

a first probe composition for hybridization to a methylated sequence of PCBD2 comprises the nucleotide sequence shown in SEQ ID NOS 109-110;

the first probe composition for hybridization with PCDHB3 methylation sequences comprises the nucleotide sequences shown as SEQ ID NOS.111-112;

the first probe composition for hybridization with PCDHGA1 methylation sequences comprises the nucleotide sequences shown as SEQ ID NOS: 113-114 (which are for hybridization with SEQ ID NO: 35) and comprises the nucleotide sequences shown as SEQ ID NOS: 115-116 (which are for hybridization with SEQ ID NO: 36);

the first probe composition for hybridization with PSORS1C2 methylation sequence comprises the nucleotide sequence shown as SEQ ID NOS.117-118;

the first probe composition for hybridization with LINC02902 methylation sequence comprises the nucleotide sequence shown in SEQ ID NOS 119-120;

The first probe composition for hybridization with a LUARIS methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS.121-122;

a first probe composition for hybridization with an ATAD2 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS.123-124;

the first probe composition for hybridization with NAT1 methylation sequence comprises a nucleotide sequence as shown in SEQ ID NOS.125-126;

a first probe composition for hybridization with the CACNA1B methylation sequence comprises the nucleotide sequence shown as SEQ ID NO:127-128 (which is used for hybridization with SEQ ID NO: 42) and comprises the nucleotide sequence shown as SEQ ID NO:129-130 (which is used for hybridization with SEQ ID NO: 43);

the first probe composition for hybridization with a UCK1 methylation sequence includes a nucleotide sequence as shown in SEQ ID NOS.131-132;

a second probe composition for hybridization to an ANKRD20A12P methylated sequence comprises the nucleotide sequence set forth in SEQ ID NOS: 133-134;

a second probe composition for hybridization to a C1orf127 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS.135-136;

the second probe composition for hybridization with a HSPA7 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS: 137-138;

A second probe composition for hybridization with ADRA2A methylation sequences comprises the nucleotide sequence shown as SEQ ID NOS.139-140;

a second probe composition for hybridization with an MTRNR2L8 methylated sequence comprises the nucleotide sequence set forth in SEQ ID NOS.141-142;

a second probe composition for hybridization to the MUS81 methylation sequence comprises the nucleotide sequence shown as SEQ ID NOS: 143-144;

the second probe composition for hybridization with the RIMBP2 methylation sequence comprises the nucleotide sequence shown as SEQ ID NOS: 145-146;

a second probe composition for hybridization with a WNK1 methylated sequence comprises the nucleotide sequence shown as SEQ ID NOS 147-148;

a second probe composition for hybridization to EDDM3A methylation sequences comprises the nucleotide sequence shown as SEQ ID NO. 149-150;

a second probe composition for hybridization with a TBPL2 methylated sequence comprises the nucleotide sequence set forth in SEQ ID NOS 151-152;

a second probe composition for hybridization with ARNT2 methylation sequence comprises the nucleotide sequence set forth in SEQ ID NOS 153-154;

a second probe composition for hybridization to MIR1282 methylation sequences comprises a nucleotide sequence as set forth in SEQ ID NOS: 155-156;

the second probe composition for hybridization with the FZD2 methylation sequence includes the nucleotide sequence shown as SEQ ID NOS.157-158;

A second probe composition for hybridization with LOC339166 methylation sequence comprises the nucleotide sequence shown as SEQ ID NOS 159-160;

a second probe composition for hybridization to ANKRD62 methylation sequences comprises the nucleotide sequence shown as SEQ ID NOS.161-162;

a second probe composition for hybridization with an ISOC2 methylation sequence comprises the nucleotide sequence set forth in SEQ ID NO. 163-164;

a second probe composition for hybridization to a LENG9 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS 165-166;

the second probe composition for hybridization with ABCG8 methylation sequences comprises the nucleotide sequences shown as SEQ ID NOS 167-168;

a second probe composition for hybridization with CDRT15P3 methylation sequences comprises the nucleotide sequence shown as SEQ ID NO. 169-170 (which is used for hybridization with SEQ ID NO. 19) and comprises the nucleotide sequence shown as SEQ ID NO. 171-172 (which is used for hybridization with SEQ ID NO. 20);

a second probe composition for hybridization to LAPTM4A methylation sequence comprises the nucleotide sequence shown in SEQ ID NOS 173-174;

a second probe composition for hybridization to ADAMTS5 methylation sequences comprises the nucleotide sequences shown in SEQ ID NOS: 175-176;

A second probe composition for hybridization to a MIR3648 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS 177-178;

a second probe composition for hybridization with RFPL2 methylation sequences comprises the nucleotide sequences shown as SEQ ID NOS: 179-180 (which are for hybridization with SEQ ID NO: 24) and comprises the nucleotide sequences shown as SEQ ID NOS: 181-182 (which are for hybridization with SEQ ID NO: 25);

a second probe composition for hybridization with the ANKRD18DP methylation sequence comprises the nucleotide sequence shown as SEQ ID NO. 183-184 for hybridization with SEQ ID NO. 26, the nucleotide sequence shown as SEQ ID NO. 185-186 for hybridization with SEQ ID NO. 27, the nucleotide sequence shown as SEQ ID NO. 187-188 for hybridization with SEQ ID NO. 28, and the nucleotide sequence shown as SEQ ID NO. 189-190 for hybridization with SEQ ID NO. 29;

a second probe composition for hybridization to FAM86HP methylation sequence comprises the nucleotide sequence shown as SEQ ID NOS 191-192;

a second probe composition for hybridization to a DUX4L8 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS 193-194;

a second probe composition for hybridization with a KIAA0825 methylation sequence comprises a nucleotide sequence shown as SEQ ID NOS 195-196;

A second probe composition for hybridization with a PCBD2 methylation sequence comprises the nucleotide sequence shown in SEQ ID NOS 197-198;

the second probe composition for hybridization with PCDHB3 methylation sequence comprises the nucleotide sequence shown as SEQ ID NOS 199-200;

a second probe composition for hybridization with a PCDHGA1 methylation sequence comprises the nucleotide sequences shown as SEQ ID NOS: 201-202 (which are for hybridization with SEQ ID NO: 35) and comprises the nucleotide sequences shown as SEQ ID NOS: 203-204 (which are for hybridization with SEQ ID NO: 36);

a second probe composition for hybridization to PSORS1C2 methylation sequence comprises the nucleotide sequence shown in SEQ ID NOS 205-206;

a second probe composition for hybridization to LINC02902 methylation sequences comprises the nucleotide sequence shown in SEQ ID NOS.207-208;

a second probe composition for hybridization with a LUARIS methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS 209-210;

a second probe composition for hybridization with an ATAD2 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS: 211-212;

a second probe composition for hybridization with NAT1 methylation sequences comprises the nucleotide sequence shown in SEQ ID NOS.213-214;

A second probe composition for hybridization with the CACNA1B methylation sequence comprises the nucleotide sequence shown as SEQ ID NO:215-216 (which is used for hybridization with SEQ ID NO: 42) and comprises the nucleotide sequence shown as SEQ ID NO:217-218 (which is used for hybridization with SEQ ID NO: 43);

a second probe composition for hybridization with a UCK1 methylation sequence includes the nucleotide sequences shown as SEQ ID NOS 219-220.

The application provides the use of a marker in the preparation of a kit for detecting colorectal cancer, wherein the gene corresponding to the marker is selected from one of the following: ANKRD20A12P, C orf127, HSPA7, ADRA2A, MTRNR L8, MUS81, RIMBP2, WNK1, EDDM3A, TBPL2, ARNT2, MIR1282, FZD2, LOC339166, ANKRD62, ISOC2, LENG9, ABCG8, CDRT15P3, LAPTM4A, ADAMTS5, MIR3648, RFPL2, ANKRD18DP, FAM86HP, DUX4L8, KIAA0825, PCBD2, PCDHB3, PCDHGA1, PSORS1C2, LINC02902, LUARIS, ATAD2, NAT1, NA1B and UCK1.

The present application provides the use of a probe composition for targeting a marker after methylation of colorectal cancer in the manufacture of a kit for detecting colorectal cancer.

In one embodiment, the probe composition is the probe composition described above.

The present application also provides a composition for colorectal cancer detection comprising a nucleic acid for detecting any methylation of the genes corresponding to the markers selected from the group consisting of: ANKRD20A12P, C orf127, HSPA7, ADRA2A, MTRNR L8, MUS81, RIMBP2, WNK1, EDDM3A, TBPL2, ARNT2, MIR1282, FZD2, LOC339166, ANKRD62, ISOC2, LENG9, ABCG8, CDRT15P3, LAPTM4A, ADAMTS5, MIR3648, RFPL2, ANKRD18DP, FAM86HP, DUX4L8, KIAA0825, PCBD2, PCDHB3, PCDHGA1, PSORS1C2, LINC02902, LUARIS, ATAD2, NAT1, NA1B and UCK1. Preferably, the nucleotide sequence of the marker is selected from one of the sequences shown in SEQ ID NOS.1-44.

In one embodiment, the nucleic acid comprises a probe composition as described above.

In one embodiment, the nucleic acid comprises:

a primer that is a fragment of at least 9 nucleotides in a target sequence of the marker, the fragment comprising at least one CpG dinucleotide sequence.

Wherein, if bisulfite is used to convert NDA in a sample to be tested, the nucleic acid for detecting methylation of the target sequence of the marker comprises a fragment of at least 9 nucleotides in the sequence after bisulfite conversion of the target sequence of the marker, said fragment comprising at least one CpG dinucleotide sequence.

In one embodiment, the nucleic acid further comprises:

a probe that hybridizes under moderately stringent or stringent conditions to at least 15 nucleotide fragments in a target sequence of the marker, the fragments comprising at least one CpG dinucleotide sequence.

In one embodiment, the composition further comprises an agent that converts the unmethylated cytosine base at position 5 of the target sequence of the marker to uracil, e.g., the agent can be bisulfite or the like; preferably, the nucleic acid for detecting methylation of a target sequence of a marker further comprises:

The blocker is used for improving the amplification specificity of the PCR amplification primer, the 5 '-end of the blocker nucleotide sequence and the 3' -end nucleotide sequence of the forward or reverse primer have an overlapping region of more than or equal to 5 nucleotides, the blocker is complementary with the forward or reverse primer and the same strand of target gene target sequence DNA, the melting temperature of the blocker is higher than that of the forward or reverse primer by more than (including) 5 ℃, and the nucleotide sequence of the blocker comprises at least one CpG dinucleotide sequence and is complementary with the sequence of the target gene target sequence DNA which is not subjected to methylation after the conversion of the bisulfite. Thus, when the genomic DNA of the biological sample to be detected is a mixture of methylated and unmethylated state, especially in the case where the DNA in the methylated state is far less than the DNA in the unmethylated state, the DNA in the unmethylated state is converted by bisulfite and preferentially binds to the blocker, and thus the DNA template binds to the PCR obligation, and thus PCR amplification does not occur, whereas the DNA in the methylated state does not bind to the blocker and thus the primer set, PCR amplification occurs, and then the fragment obtained by the amplification is detected directly or indirectly.

The present application provides a kit comprising reagents for detecting the above-described markers or the above-described probe composition or the above-described composition.

In one embodiment, the kit further comprises a container for holding a biological sample of a subject.

In one embodiment, the kit further comprises instructions for use and interpretation of the test results.

The biological sample may be, for example, peripheral blood whole blood, plasma or serum.

The method for detecting the methylation level of a target sequence using the above-described kit is not limited in any way, and one skilled in the art can select as desired, for example, the present application provides a method for detecting the methylation level of a marker target sequence using the above-described kit, which comprises the steps of:

collecting a sample of a subject;

extracting and purifying DNA in the sample;

constructing a DNA library for sequencing against the purified DNA sample;

transforming said constructed DNA library with bisulfite;

pre-PCR amplifying the bisulfite converted DNA library;

performing hybridization capture on the sample amplified by the pre-PCR by using the probe composition;

amplifying the hybridized and captured product by utilizing PCR;

Performing high-throughput second-generation sequencing on the PCR amplified product after hybridization capture;

analyzing the sequencing data to determine the methylation level of the sample;

calculating a threshold value for each marker based on methylation of an existing sample, interpreting the patient's disease based on the methylation level of a certain marker of the sample, if the methylation level of a certain marker of the sample exceeds the threshold value, it is a cancer sample, if it is below the threshold value, it is a healthy human sample.

Also for example, the present application provides a method for detecting the methylation level of a target sequence of a marker using the kit described above, comprising the steps of:

(1) Extracting peripheral blood of a subject, and separating plasma or serum;

(2) Extracting free DNA from plasma or serum;

(3) Treating the free DNA obtained in step (2) with a reagent to convert the unmethylated cytosine base at position 5 to uracil or other bases, i.e., to convert the unmethylated cytosine base at position 5 of the target sequence of the marker to uracil or other bases, the converted bases differing from the unmethylated cytosine base at position 5 in hybridization performance and being detectable;

(4) Contacting the free DNA treated in step (3) with a DNA polymerase and primers for the target sequence of the marker such that the target sequence of the treated marker is amplified to produce amplified products or not amplified; the target sequence of the treated marker, if subjected to DNA polymerization, produces amplification products; the target sequence of the treated marker is not amplified if DNA polymerization does not occur;

(5) Detecting the amplified product with a probe;

(6) Determining the methylation status of at least one CpG dinucleotide of the target sequence of the marker based on the presence or absence of the amplification product, thereby determining the methylation level of the target sequence of the marker.

The present application provides a chip comprising the above-described marker or the above-described probe composition or the above-described composition.

The sequencing principle of the chip, also called a gene chip, is a hybridization sequencing method, namely a method for determining the sequence of nucleic acid by hybridizing with a group of nucleic acid probes with known sequences, wherein probes with target nucleotides with known sequences are immobilized on the surface of a substrate. When the nucleic acid sequence with fluorescent mark in the solution is complementarily matched with the nucleic acid probe at the corresponding position on the gene chip, a group of probe sequences with complete complementation of the sequences are obtained by determining the probe position with the strongest fluorescence intensity.

The chip is prepared by mainly taking a glass sheet or a silicon wafer as a carrier, and sequentially arranging oligonucleotide fragments or cDNA (complementary deoxyribonucleic acid) serving as probes on the carrier by adopting an in-situ synthesis and microarray method.

The chip is based on signal detection of DNA sequence hybridization after bisulfite treatment, wherein unmethylated cytosine is changed into uracil, methylated cytosine is kept unchanged, uracil is converted into thymine, and finally chip hybridization is carried out; finally, judging the type of the added base according to the fluorescence color, and further determining whether the locus is methylated.

The present application provides a method of colorectal cancer screening comprising:

detecting methylation level of a marker

Determining the risk of the subject for colorectal cancer based on the methylation level, wherein the gene corresponding to the marker is selected from one of the following: ANKRD20A12P, C orf127, HSPA7, ADRA2A, MTRNR L8, MUS81, RIMBP2, WNK1, EDDM3A, TBPL2, ARNT2, MIR1282, FZD2, LOC339166, ANKRD62, ISOC2, LENG9, ABCG8, CDRT15P3, LAPTM4A, ADAMTS5, MIR3648, RFPL2, ANKRD18DP, FAM86HP, DUX4L8, KIAA0825, PCBD2, PCDHB3, PCDHGA1, PSORS1C2, LINC02902, LUARIS, ATAD2, NAT1, NA1B and UCK1.

Examples

The materials used in the test and the test methods are generally and/or specifically described herein, and in the examples which follow,% represents wt%, i.e., weight percent, unless otherwise specified. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.

Example 1

Screening markers

1) Sample collection: a total of 58 plasma samples were collected from cooperating hospitals for colorectal cancer patients, and 42 healthy human plasma samples were collected. The following experiment was performed (see 1.1 for details).

2) Candidate marker analysis: differential analysis of healthy and colorectal cancer patients was performed using DSS software for methylation sites derived from sequencing data. Criteria for filtration: p <0.05 and the difference between the two sets of means is greater than 0.1. 561 differential methylation regions were finally obtained.

3) And (3) marker verification: probes were designed for capture of 561 different methylation regions, and the results were verified using bohr plasma sample data (colorectal cancer sample number=38, healthy person sample number=28) to obtain 44 markers that can distinguish colorectal cancer from healthy persons. The sequences are shown as SEQ ID NO. 1-SEQ ID NO. 44.

Based on the resulting target sequence region, a probe composition (panel) is tailored.

Then validated in plasma samples, qiagen was used in the experimentsCirculating Nucleic Acid Kit cfDNA preparation was performed with a product number 55114. ACCEL- & lt- & gt using shift>The preparation of the library was performed with METHYL-SEQ DNA LIBRARY KIT, product number 30096, and the experimental detection method was as follows,

cfdna extraction purification:

1.1.1. plasma sample preparation:

the blood samples were centrifuged at 2000g for 10min at 4℃and the plasma was transferred to a new centrifuge tube. The plasma samples were centrifuged at 16000g for 10min at 4℃and the next step was performed depending on the type of collection tube used, which was the other one used in the experiment.

1.1.2. Cleavage and binding:

1.1.2.1. mu.l, 200. Mu.l, 300. Mu.l, 400. Mu.l, 500. Mu.l QIAGEN proteinase K were pipetted into 50ml centrifuge tubes.

1.1.2.2. 1ml, 2ml, 3ml, 4ml, 5ml plasma or serum was added to the 50ml centrifuge tube.

1.1.2.3. 0.8ml, 1.6ml, 2.4ml, 3.2ml, 4.0ml Buffer ACL (containing 1.0. Mu.g carrier RNA) was added, capped and vortexed for 30s; note that: fully and uniformly mixing to ensure full cracking; the next step is immediately performed.

1.1.2.4. Incubate at 60℃for 30min.

1.1.2.5. Taking out the centrifuge tube, placing the centrifuge tube on a test bed, and unscrewing the tube cover.

1.1.2.6. 1.8ml, 3.6ml, 5.4ml, 7.2ml, 9.0ml Buffer ACB was added to a 50ml centrifuge tube; covering the cover, and mixing for 15-30s.

1.1.2.7. The lysis mixture was incubated on ice for 5min.

1.1.2.8. QIAampMini column was inserted onto the vacuum pump adapter and 20ml tube extension was inserted onto the column; and (3) injection: ensure that tube extender is firmly inserted on QIAamp Mini column, avoid sample leakage.

1.1.2.9. The lysate-buffer ACB mixture from step 7 was carefully added to the tube extension of QIAamp Mini column and the vacuum pump was turned on; after all lysates were completely withdrawn from Mini column, the vacuum pump was turned off and the pressure was released to 0mbar; the expander is carefully removed and discarded.

1.1.3. Washing:

1.1.3.1. adding 600 μl Buffer ACW1 to Mini column, opening the cover, and starting the vacuum pump; after all liquid had passed the column membrane, the vacuum pump was turned off and the pressure was released to 0mbar.

1.1.3.2. Adding 750 μl Buffer ACW2 into Mini column, opening the cover, and starting vacuum pump; after all liquid had passed the column membrane, the vacuum pump was turned off and the pressure was released to 0mbar.

1.1.3.3. Adding 750 μl ethanol (96-100%) into Mini column, opening the cover, and turning on vacuum pump; after all liquid had passed the column membrane, the vacuum pump was turned off and the pressure was released to 0mbar.

1.1.3.4. The VacConnector is discarded by capping QIAamp Mini column and removing it from the adapter. QIAamp Mini column was placed in a clean 2ml collection tube and centrifuged at high speed (20000 g;14000 rpm) for 3min.

1.1.3.5. QIAamp Mini column was placed in a new 2ml collection tube. The tube lid was opened and incubated at room temperature for 5min to allow the membrane to dry completely.

1.1.4. Eluting cfDNA:

1.1.4.1. QIAamp Mini column was placed in a 1.5ml elution tube and the 2ml collection tube from step 14 was discarded. Adding 20-150 μl Buffer AVE to the center of Mini membrane; the tube was covered and incubated for 3min at room temperature.

1.1.4.2. The nucleic acids were eluted by centrifugation at full speed (20000 g;14000 rpm) for 1min in a centrifuge.

For cfDNA samples, agilent2100 performed fragment detection, and direct Qubit was used for subsequent experiments.

Bisulfite conversion and purification:

1.2.1. prepare CT Conversion Reagent:

1.2.1.1. 700. Mu.l NF water, 300. Mu. l M-division Buffer and 50. Mu. l M-Dissolving Buffer were added to a tube of CT transforming reagent, mixed well at room temperature, and vortexed or shaken frequently for 10min.

1.2.1.2. And (5) after mixing uniformly, split charging, and preparing the mixture into 10 reaction quantities once.

1.2.2. The DNA library was subjected to bisulfite conversion, and a reaction system was prepared according to the following table.

TABLE 1

1.2.3. The pipette was adjusted to 100. Mu.l, gently blotted and mixed 6 times, then split into two tubes and placed on a PCR instrument.

1.2.4. The following procedure was set up for the reaction on the PCR instrument: the thermal head temperature was 105 ℃.

TABLE 2

Temperature (temperature)	Time
		98℃	10min
64℃	2.5h
		4℃	∞

1.2.5. A new 1.5ml centrifuge tube was taken and 600. Mu.l of M-Binding Buffer was added.

After the PCR was completed, the two identical samples were transferred to the corresponding 1.5ml centrifuge tubes by brief centrifugation, and mixed well.

1.2.7. The above mixed sample was added to the Zymo-SpinTMIC Column, mixed upside down, and centrifuged at 10,000Xg for 30s.

1.2.8. 100. Mu.l of M-Wash Buffer was added to the column and centrifuged at 10,000Xg for 30s.

1.2.9. 200. Mu.l of M-Desulphonation Buffer was added to the column, and the mixture was allowed to stand at room temperature for 15 to 20 minutes and centrifuged at 10,000Xg for 30 seconds.

1.2.10. 200 μl of M-Wash Buffer was added to the column and centrifuged at 10,000Xg for 30s.

1.2.11. The above steps are repeated once.

1.2.12. The column was placed in a new collection tube and centrifuged again at 10,000Xg for 30s.

1.2.13. The recovery column was placed in a new 1.5ml EP tube, 15. Mu.l of LOW EDTA buffer was added to the center of the column membrane and centrifuged at 10,000Xg for 30s.

1.3. Denaturation:

1.3.1. the PCR instrument was preheated to 95 ℃.

1.3.2. The following procedure was set up for the reaction on the PCR instrument: the thermal head temperature was 105 ℃.

TABLE 3 Table 3

Temperature (temperature)	Time
		95℃	∞
95℃	2min
		95℃	∞

1.3.3. Immediately after incubation was completed, the tubes were placed on ice for 2min.

1.4. Joint connection and purification:

1.4.1. the reaction system was configured with reference to the following table:

TABLE 4 Table 4

1.4.2. The following procedure was set up for the reaction on the PCR instrument: the thermal head temperature was 105 ℃.

TABLE 5

Temperature (temperature)	Time
		37℃	∞
37℃	15min
		95℃	2min
4℃	∞

1.5. Sample extension and purification:

1.5.1. the reaction system was configured with reference to the following table:

TABLE 6

Component (A)	Volume of
		Reagent Y1	2ul
Enzyme Y2	42ul
		totalVolume	44ul

1.5.2. The following procedure was set up for the reaction on the PCR instrument: the thermal head temperature was 105 ℃.

TABLE 7

Temperature (temperature)	Time
		98℃	∞
98℃	1min
		62℃	2min
65℃	5min
		4℃	∞

The DNA protection buffer was added to the liquid to turn blue. Gently blotted and mixed, and then split into two tubes for PCR.

1.5.4. The following procedure was set up and run: the lid was heated to 105 ℃.

TABLE 8

Temperature (temperature)	Time
		95℃	5min
60℃	10min
		95℃	5min
60℃	10min
		4℃	∞

1.5.5. The purification system was prepared according to the following table:

TABLE 9

1.5.6. And adding the magnetic beads with the proportion into each sample for recycling, and vibrating and mixing uniformly to instantaneously separate.

1.5.7. And standing at room temperature for 5min.

1.5.8. Shaking, mixing, centrifuging, placing on a magnetic rack, adsorbing until the solution is about 2min, and sucking the supernatant after the solution is clear.

1.5.9. Mu.l of 80% ethanol was added to wash the beads for 30s, the supernatant was discarded, and all the remaining ethanol on the inner wall of the dropper was carefully removed therefrom.

1.5.10. Repeating the above steps.

1.5.11. Adding the optimal volume of low EDTA TE buffer solution recommended in the table, eluting, and shaking and mixing.

1.5.12. Incubate for 2min at room temperature.

1.5.13. Placing on a magnetic rack for adsorption until the solution is clear (about 2 min), and sucking the supernatant after the solution is clear.

1.5.14. The whole eluate was transferred to a new 0.2mL PCR tube, ensuring that the eluate did not contain magnetic beads.

1.6. Joint connection and purification:

1.6.1. library reaction systems were prepared according to the following table:

table 10

1.6.2. The following procedure was set up and run: thermal cover 0 deg.c:

TABLE 11

Temperature (temperature)	Time
		25℃	∞
25℃	15min
		4℃	∞

1.6.3. The purification system was prepared according to the following table:

table 12

1.6.4. And adding the magnetic beads with the proportion into each sample for recycling, and vibrating and mixing uniformly to instantaneously separate.

1.6.5. And standing at room temperature for 5min.

1.6.6. Shaking, mixing, centrifuging, placing on a magnetic rack, adsorbing until the solution is about 2min, and sucking the supernatant after the solution is clear.

1.6.7. Mu.l of 80% ethanol was added to wash the beads for 30s, the supernatant was discarded, and all the remaining ethanol on the inner wall of the dropper was carefully removed therefrom.

1.6.8. Repeating the above steps.

1.6.9. Adding the optimal volume of low EDTA TE buffer solution recommended in the table, eluting, and shaking and mixing.

1.6.10. Incubate for 2min at room temperature.

1.6.11. Placing on a magnetic rack for adsorption until the solution is clear (about 2 min), and sucking the supernatant after the solution is clear.

1.6.12. The whole eluate was transferred to a new 0.2mL PCR tube, ensuring that the eluate did not contain magnetic beads.

1.7. Library amplification and purification:

1.7.1. library reaction systems were prepared according to the following table:

TABLE 13

1.7.2. The following procedure was set up and run: thermal cover 105 deg.c:

TABLE 14

1.7.3. The recommended cycle numbers are as follows:

TABLE 15

Input amount	Number of recommended cycles
		20ng cfDNA	10-11
100ng gDNA	9-10
		20ng gDNA	11-12

1.7.4. The purification system was prepared according to the following table:

table 16

1.7.5. The PCR product was transferred to a 1.5ml centrifuge tube.

1.7.6. And adding the magnetic beads with the proportion into each sample for recycling, and vibrating and mixing uniformly to instantaneously separate.

1.7.7. And standing at room temperature for 5min.

1.7.8. Shaking, mixing, centrifuging, placing on a magnetic rack, adsorbing until the solution is about 2min, and sucking the supernatant after the solution is clear.

1.7.9. Mu.l of 80% ethanol was added to wash the beads for 30s, the supernatant was discarded, and all the remaining ethanol on the inner wall of the dropper was carefully removed therefrom.

1.7.10. Repeating the above steps.

1.7.11. The magnet rack is left for 5-10 minutes until the beads are dried (avoiding excessive drying, which may lead to reduced DNA recovery).

1.7.12. Adding the optimal volume of low EDTA TE buffer solution recommended in the table, eluting, and shaking and mixing.

1.7.13. Incubate for 2min at room temperature.

1.7.14. Placing on a magnetic rack for adsorption until the solution is clear (about 2 min), and sucking the supernatant after the solution is clear.

1.7.15. The whole eluate was transferred to a new 0.2mL PCR tube, ensuring that the eluate did not contain magnetic beads.

1.7.16. Aspirate 1ul for qubit calibration and perform 2100 quality checks.

1.8. Hybridization of sample with probe:

1.8.1. mixing samples:

the amounts of DNA library used are referred to in the following table, and the total amount may be in excess of 1500ng total, but no more than 4ug;

TABLE 17

1.8.1.2. Calculating the dosage of different samples, and uniformly mixing in a centrifuge tube.

1.8.1.3. The following prehybridization reagents are added to the mixed samples, and the mixture is uniformly mixed, so that bubbles are not generated as much as possible.

TABLE 18

1.8.1.4. And (3) drying the mixed prehybridization reagent in a vacuum concentrator at normal temperature (if heating is needed, the low temperature is needed).

1.8.2. Hybridization:

1.8.2.1. fast Hybridization Mix is incubated at 65℃for 10min or until all the pellet is dissolved, vortexed rapidly and added to 20ul of the sample lyophilized in the previous step to resuspend the sample (please not return the hybridization solution to room temperature), and the fingertips flick to mix well to avoid air bubbles.

1.8.2.2. Air bubbles were removed by rapid centrifugation and 30ul Hybridization Enhancer a was added to the above reagent surface.

1.8.2.3. The PCR tube was placed in a preheated PCR instrument for hybridization.

1.8.2.4. The following procedure was set up and run: the lid was heated to 85 ℃.

TABLE 19

Temperature (temperature)	Time
		95℃	∞
95℃	5min
		60℃	15min-4h

1.8.3. Combining:

1.8.3.1. shake pre-equilibrated streptavidin beads until complete mixing, add 100 μl of beads to a 1.5ml centrifuge tube.

1.8.3.2. 200ul of binding buffer was added and mixed by blowing with a gun head.

1.8.3.3. Placing the centrifuge tube on a magnetic rack for 1min or clarifying the solution, discarding the supernatant, and taking down the centrifuge tube.

1.8.3.4. The above washing steps were repeated 2 times for a total of 3 times.

1.8.3.5. After the last wash, 200ul of binding buffer was added and resuspended with shaking to allow adequate mixing.

1.8.3.6. After hybridization, the lid of the PCR instrument was opened and the hybridization solution was rapidly transferred to the well-equilibrated magnetic beads.

1.8.3.7. The magnetic beads to which the hybridization solution was added were thoroughly mixed on a Shaker, a rotator or a rotator at room temperature for 30 minutes.

1.8.3.8. Taking down the centrifuge tube from the mixing instrument, placing the centrifuge tube on a magnetic rack for 1min after quick centrifugation, removing the supernatant, and taking down the tube.

1.8.3.9. 200ul of preheated washing liquid 1 was added and mixed well.

Incubation was carried out at 1.8.3.10.63 ℃or 65℃for 5min.

1.8.3.11. The centrifuge tube was placed on a magnetic rack for 1min, the supernatant was removed, and the tube was removed.

1.8.3.12. Repeating the above steps, adding 200ul of preheated washing liquid 1 again, and mixing uniformly.

1.8.3.13. Incubation was performed at 63℃or 65℃for 5min.

1.8.3.14. Transferring the liquid to a new tube; placing on a magnetic rack for 1min, removing supernatant, and taking down the tube.

1.8.3.15. 200ul of preheated wash buffer 2 is added and the gun head is mixed uniformly.

Incubation was carried out at 1.8.3.16.48 ℃for 5min.

1.8.3.17. Placing on a magnetic rack for 1min, removing supernatant, and taking down the tube.

1.8.3.18. The washing was repeated (steps 3.15-3.17) 2 times for a total of three times.

1.8.3.19. Finally, the wash was blotted with a 10ul gun head.

1.8.3.20. 45ul of water was added, mixed well and the solution incubated on ice.

1.8.4. Post-capture PCR amplification, purification and quality control:

1.8.4.1. the following procedure was set up and run: the lid was heated to 105 ℃.

Table 20

1.8.4.2. Mix the bead mixture in 1.3 and aspirate 22.5ul to 0.2ml PCR tube.

1.8.4.3. 2.5ul of amplification primers, 25ul KAPA HiFi HotStart ReadyMix, and a total of 50ul of reaction system were added to a 0.2ml PCR tube.

1.8.4.4. Mixing with gun head, centrifuging, and placing into PCR instrument to start amplification.

1.8.4.5. Vortex thoroughly mix pre-equilibrated DNA purification beads.

1.8.4.6. 90ul (1.8 x) of DNA purification magnetic beads are added to the amplified PCR product, and the mixture is well mixed by vortex.

1.8.4.7. Incubate at room temperature for 5min.

1.8.4.8. The centrifuge tube was placed on a magnetic rack for 1min and the supernatant was removed after the solution was clear.

1.8.4.9. Directly adding 200ul of 80% ethanol which is prepared at present without taking down the centrifuge tube from the magnetic rack, incubating for 1min, and discarding the supernatant; the 80% ethanol wash was repeated once (2 times total) and the centrifuge tube was kept on a magnet rack.

1.8.4.10. The remaining ethanol was carefully removed with a 10ul gun head and left at room temperature for 5-10min or until the beads were dried, taking care not to overdry the beads.

1.8.4.11. Taking off the tube from the magnetic rack, adding 32ul of water, blowing with gun head, mixing thoroughly, incubating at room temperature for 2min

1.8.4.12. The centrifuge tube was placed on a magnetic rack for 3min or until the solution was clear.

1.8.4.13. Transfer 30ul of supernatant to a clean 0.2ml centrifuge tube.

1.8.4.14. 1 μl of library was quantified using Qubit and library concentrations were recorded.

1.8.4.15. 1 μl of sample was taken and used for library fragment length determination using Agilent 2100.

1.8.4.16. Sequencing was performed using Illumina high throughput sequencing platform.

1.9. Methylation letter analysis flow. The method is approximately as follows: checking sequencing quality by using fastp quality control software, removing low-quality reads, comparing the quality-controlled clean data to a reference genome by using Bismark comparison software, and extracting corresponding methylation sites by using bismark_methyl_extraction software. Finally, the methylation level of the target region is calculated, and the value results in a diagnosis of cancer if the threshold value is exceeded and a diagnosis of normal if the threshold value is lowered.

Example 2

The present example discloses a methylation specific biomarker for diagnosing colorectal cancer, based on the data set of 58 colorectal cancer samples and 42 normal human samples, the methylation library building method described in the example 1 is used to screen out biomarkers related to colorectal cancer by using methylation level differences in different groups (colorectal cancer and normal), and the independent data sets of the site data in normal and colorectal cancer samples are verified, and 44 DNA fragments most notably distinguishing normal and cancer samples are screened out altogether, wherein the 44 methylation biomarkers (sites or markers for short) and the discrimination threshold are shown in table 21.

The methylation level threshold calculation method comprises the following steps: drawing an ROC curve according to the data set (comprising the type and methylation level of each sample), wherein the confusion matrix corresponding to the optimal threshold point on the ROC curve is the basis for calculating indexes such as sensitivity, specificity and accuracy. Typically we will choose by a boulder index (you index). The about index, also called the correct index, refers to the sum of sensitivity and specificity minus 1: about index = sensitivity + specificity-1. The value of about dengue index range is between 0 and 1, which represents the total ability of the classification model to find true patients and non-patients. The greater the about log index, the better the classification model performance, the sensitivity and specificity of each marker is shown in table 21. As can be seen from table 21, the AUC values for the markers described herein are higher.

TABLE 21 characterization data for 44 methylation markers

Example 3

Peripheral blood was collected as in example 1 using the methylation marker detection method of the present application using 6 additional collected human samples (S1-3 is a healthy human sample and S4-6 is a colorectal cancer patient sample); establishing a library, and sequencing through an Illumina platform; sequencing data is subjected to the biological information analysis flow to obtain the methylation level of each marker, and according to the threshold value of each marker, the disease condition of the patient is predicted, if the threshold value is exceeded, a cancer sample is obtained, and if the threshold value is lower than the threshold value, a healthy human sample is obtained, (SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.21, SEQ ID NO.26-SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.33 and SEQ ID NO.37 are more specific, and if the threshold value is exceeded, a healthy human sample is obtained, and if the threshold value is lower than the threshold value, a cancer sample is obtained), and specific results are shown in the following table 22:

wherein, the interpretation result, 0, represents the classification as normal, i.e. healthy; 1 represents a classification as abnormal, i.e. tumor.

Table 22 methylation values and interpretation results for the samples

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In summary, the inventors of the present application have obtained methylation genes related to colorectal cancer, and determined target sequences of methylation abnormality of colorectal cancer methylation genes, and through the target sequences of these methylation genes, the methylation state of the genes can be sensitively and specifically detected, so that the methylation state can be used for detecting free DNA of peripheral blood, and the composition described in the present application can realize real-time monitoring, with higher sensitivity and accuracy.

The above description is only of the preferred embodiments of the present application and is not intended to limit the present application in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present application still fall within the protection scope of the technical solution of the present application.

The sequence table used in the application is shown in table 23:

table 23

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Claims

2. The marker according to claim 1, wherein the nucleotide sequence of the marker is selected from one of the markers shown in SEQ ID NOs 1-44, preferably the marker is a methylated marker.

3. A probe composition comprising a probe that targets methylation of a marker according to claim 1 or 2.

4. A probe composition according to claim 3, wherein the probe composition comprises a hypermethylated first probe composition for hybridization to a bisulfite converted hypermethylated region and a hypomethylated second probe composition for hybridization to a bisulfite converted hypomethylated region;

preferably, n and m are each any integer from 1 to 10;

preferably, there is x between the n-1 th probe and the n-th probe ₁ Overlapping of nucleotides, preferably x ₁ Is any integer from 0 to 100;

6. The use according to claim 5, wherein the nucleotide sequence of the marker is selected from one of the markers shown in SEQ ID NOs 1-44, preferably the marker is a methylated marker;

preferably, the probe composition is the probe composition of claim 3 or 4.

8. The composition of claim 7, wherein the nucleotide sequence of the marker is selected from one of the nucleotide sequences set forth in SEQ ID NOS.1-44.

9. The composition of claim 7 or 8, wherein the nucleic acid comprises the probe composition of claim 3 or 4;

preferably, the nucleic acid comprises:

preferably, the nucleic acid further comprises:

10. Kit, characterized in that it comprises a reagent for detecting a marker according to claim 1 or 2 or a probe composition according to claim 3 or 4 or a composition according to any one of claims 7 to 9.

11. Chip, characterized in that it comprises a marker according to claim 1 or 2 or a probe composition according to claim 3 or 4 or a composition according to claim 7 or 8.