Method and combination for one-tube simultaneous detection of tumor-specific gene mutation and methylation, and use thereof

Abstract

Provided are a method and a combination for one-tube simultaneous detection of tumor-specific gene mutation and methylation, and the use thereof. The method comprises searching a database to select mutation and methylation sites of a cancer driver gene and a cancer suppressor gene, synthesizing capture probes correspondingly paired with a target interval, performing hybrid capture on an amplification product of a sample treated with a restriction endonuclease by using a probe set comprising tumor-specific gene mutation and methylation capture probes, constructing a high-throughput sequencing library containing a target region, and performing high-throughput sequencing to obtain the sequence of a target fragment, so as to obtain the results of methylation, point mutations and indel variation of a cancer-associated gene. The provided method enables one-tube simultaneous detection of mutation and methylation states of a cancer-associated gene in one assay, requires a low content of a gene to be detected, can be used for methylation and mutation detection of a tumor-specific DNA, and has low detection costs and high efficiency.

Description

A method, combination and application for simultaneously detecting tumor-specific gene mutation and methylation in one tube

[0001] Cross-reference to Related Applications

[0002] This application claims priority to the Chinese patent application No. 202411242027.1, filed on September 05, 2024, and entitled "A method, combination and application for simultaneously detecting tumor-specific gene mutation and methylation in one tube", the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application belongs to the technical field of gene detection. Specifically, the present application provides a method and reagent for simultaneously detecting tumor-specific gene mutation and methylation in one tube. BACKGROUND

[0004] Cancer is one of the main causes of human death, and the main limiting factor affecting the clinical efficacy of cancer is the period of discovery and diagnosis. If diagnosed and removed at an early stage (before cancer cell metastasis), cancer can achieve a cure. Early screening and early diagnosis of cancer is an important way to reduce cancer mortality.

[0005] Early detection of cancer is very important for treatment effect, but it is also a difficult point that has been difficult to overcome in the field. Currently, early screening methods for tumors in clinical practice include imaging, endoscopy, and tumor markers. These detection techniques have defects such as invasiveness, discomfort during screening, and low detection sensitivity. Traditional methods have various limitations, and there is an urgent need for early screening techniques with high sensitivity / specificity and convenient operation in clinical practice.

[0006] In recent years, DNA molecular detection technology has developed rapidly, and tumor molecular marker detection has become a new field of tumor molecular diagnosis following imaging diagnosis and pathological diagnosis. Tumor DNA markers can be detected in body fluids, which can reflect the presence of tumors, malignancy, and have important significance for prognosis estimation and treatment effect evaluation.

[0007] Liquid biopsy as a new non-invasive detection technology has great application potential in early screening of tumors. Tumor markers for liquid biopsy include genome, epigenome, transcriptome, microbiome, proteome, and metabolome. The characteristics of tumor markers in the ctDNA layer include point mutations, fragmentation, copy number changes, and methylation. Among them, DNA mutation and methylation occur in almost all tumors. Therefore, the combination of mutation and methylation co-detection technology can help the landing of multi-cancer early screening technology.

[0008] One of the gold standards of methylation sequencing is bisulfite sequencing, abbreviated as BS-seq. Its detection principle is to treat the sample DNA with bisulfite, convert the unmethylated C base into U base, and then amplify it by PCR. The U base is converted into T, and the sequence is compared with the unconverted sequence to determine whether methylation modification occurs at this site. This method has a high degradation rate of DNA, and cannot achieve the purpose of co-detection of mutations and methylation in the same hybrid capture system, so it cannot simultaneously detect methylation and mutations.

[0009] The existing gene detection method is mostly focused on separately detecting mutations and methylation states, and there are few reports on simultaneously detecting multiple cancer gene mutations and methylation states. The limitations of clinical detection range (mutation or methylation detection of a few sites), the low content of ctDNA commonly used in liquid biopsy (mutation frequency is generally as low as 0.1-1%), and the high detection cost of Sanger sequencing technology for multi-gene detection greatly limit its practical clinical application. Therefore, it is an urgent problem to establish a high-efficiency, fast, sensitive, and economical method for simultaneously detecting multiple cancer gene mutations and methylation states in one tube. SUMMARY

[0010] In one aspect, the present application provides a method for simultaneously detecting tumor-specific gene mutations and methylation in one tube, which comprises the following steps:

[0011] (1) selecting tumor-specific mutant genes and tumor-specific methylation genes, selecting mutation sites of tumor-specific mutant genes and methylation sites of tumor-specific methylation genes, and selecting regions of 30-100 bp upstream and downstream of the mutation sites of tumor-specific mutant genes and the methylation sites of tumor-specific methylation genes as tumor-specific mutant gene target intervals and tumor-specific methylation gene target intervals;

[0012] (2) synthesizing target interval corresponding paired capture probes, which are composed of probe combination sequences complementary to adjacent capture probes in the same interval and target-specific sequences complementary to tumor-specific mutant gene target intervals or tumor-specific methylation gene target intervals;

[0013] (3) extracting free DNA or genomic DNA from the sample to be detected, mixing with exogenous DNA, end-repairing, adding "A", and ligating, and obtaining sample ligation products of the sample after purification;

[0014] (4) treating the ligation products of the sample with methylation-sensitive restriction endonuclease, connecting index adapters to the DNA fragments by PCR amplification, and obtaining sample DNA methylation and mutation pre-library after purification;

[0015] (5) hybridizing the DNA methylation and mutation pre-library in step (4) with a capture probe to capture the methylation and mutation DNA sequences of the target region;

[0016] (6) PCR amplifying the DNA sequences of the target region to obtain an amplification product;

[0017] (7) purifying the amplification product in step (6) and obtaining the methylation and mutation library of the target region after quality inspection;

[0018] (8) high-throughput sequencing the methylation and mutation library of the target region constructed in step (7) to obtain the sequence of the target fragment;

[0019] (9) comparing the sequence information of the target fragment obtained with the normal gene sequence to obtain the mutation and methylation information of the target fragment.

[0020] The interval described in the present application refers to a specific part of a specific gene to be detected, for example, the "H4C6 gene chr6:26240658-26240991 position" shown in the examples.

[0021] Further, the methylation and mutation sites of the cancer-related genes are selected in step (1) in combination with literature and TCGA and COSMIC data.

[0022] Further, the tumor-specific mutation gene is selected from one or more of NRAS, CTNNB1, PIK3CA, FBXW7, APC, EGFR, BRAF, CDKN2A, PTEN, FGFR2, HRAS, KRAS, AKT1, TP53, PPP2R1A, and GNAS.

[0023] Further, the tumor-specific mutant gene target intervals are chr1:115256462-115256611, chr1:115258679-115258834, chr10:89692723-89693040, chr10:123279607-123279757, chr11:534208-534345, chr12:25378488-25378637, chr12:25380204-25380355, chr12:25398219-25398366, chr14:105246490-105246643, chr17:7572893-7573080, chr17:7573907-7574089, chr17:7576783-7577228, chr17:7577440-7577667, chr17:7578099-7578633, chr17:7579220-7579944, chr19:52715900-52716054, chr20:57484357-57484503, chr3:41266034-41266205, chr3:178916784-178916941, chr3:178921480-178921626, chr3:178936016-178936175, chr3:178951998-178952156, chr4:153247221-153247419, chr4:153249298-153249453, chr4:153251828-153251987, chr5:112175138-112175286, chr5:112175578-112175730, chr7:5571691-5571899, chr7:55232932-55233170, chr7:55241573-55241776, chr7:55242374-55242553, chr7:55248945-55249211, chr7:55259371-55259607, chr7:55268840-55269073, chr7:140453072-140453231, chr9:21971056-21971281.

[0024] Further, the tumor specific methylation genes are SHOX2, PTGER4, H4C6, RASSF1A, Septin9, RNF180, SDC2, PAX1, ZNF671, BMPR1A, PLAC8, EVX1, GNB4, SIX6, PCDHGB7, DAPK1, GSTP1, HIC1, RUNX3, VHL, WT1, BMP3, NDRG4, BCAT1, Twist1, JAM3, TFPI2, CDO1, LYPD5, HOXA9, BNC1, PENK, SFRP1, PCDH8, ADAM23, CHST11, C2orf88, MCF2L, CAMK2N1, TACSTD2.

[0025] Tumor-specific methylation gene target intervals are chr1 :20810296-20810635, chr1 :25255411-25255652, chr1 :59042517-59043026, chr10:88515764-88516078, chr10:88516143-88516235, chr11 :32354800-32355439, chr11 :67350903-67351488, chr11 :133939397-133939638, chr12:25055729-25056569, chr12:104850537-104850822, chr13:53421948-53422217, chr13:53422689-53422828, chr13:113623367-113623449, chr13:113623511-113623603, chr14:60976415-60976602, chr15:83952782-83953090, chr16:58497189-58497988, chr17:1957373-1957754, chr17:75368649-75370546, chr19:44302727-44303241, chr19:58238546-58239068, chr2:191044925-191045141, chr2:207308920-207309240, chr20:21686160-21686478, chr20:21686644-21687729, chr3:10183433-10183998, chr3:50377764-50378580, chr3:157821193-157821570, chr3:179168719-179168984, chr3:179169053-179169633, chr4:81951952-81952848, chr4:84030745-84031349, chr5:40680835-40682071, chr5:63461409-63461789, chr5:63461875-63462146, chr5:115151476-115152553, chr5:140797123-140797741, chr6:26240658-26240991, chr7:19147401-19147925, chr7:27204944-27205261, chr7:27282047-27282879,chr7:27282942-27283176, chr7:93519247-93520492, chr8:41166830-41167127, chr8:41168404-41168548, chr8:57358291-57359351, chr8:97505708-97507647, chr9:90113255-90113681.

[0026] Further, the mutation site of the variation gene type includes point mutations and insertion-deletion variations.

[0027] Further, the methylation site includes a site recognized by a methylation-sensitive restriction enzyme.

[0028] Further, the methylation-sensitive restriction enzyme is selected from the group consisting of HpaI, HpaII, HhaI, AciI, and combinations thereof.

[0029] Further, the 5' end of the capture probe is a first probe binding sequence, which is complementary to the 3' end of another capture probe 1, and the 3' end of the capture probe is a second probe binding sequence, which is complementary to the 5' end of another capture probe 2; the length of the first and second probe binding sequences is 8-30 nt, the length of the target-specific sequence is 20-80 nt, the 5' end of the capture probe is biotin-modified; the other capture probe 1 and the other capture probe 2 are not the same probe.

[0030] Further, in step (3), genomic DNA is extracted from a cell line, tumor tissue or exfoliated cells, or free DNA is extracted from plasma, urine, pleural effusion or ascites.

[0031] Further, in step (3), the sample ligation product includes all sequences of the sample DNA and the added exogenous DNA.

[0032] Further, the exogenous DNA is Lambda DNA, which is used to monitor the efficiency of enzyme digestion of the methylation-sensitive restriction enzyme.

[0033] Further, in step (5), 1-10 sample pre-libraries are hybridized simultaneously.

[0034] Further, the library concentration required for the outbound quality inspection is greater than or equal to 1.5 ng / μL, and the average fragment distribution is 200-1000 bp.

[0035] The above method can be used for diagnostic or non-diagnostic purposes, including but not limited to scientific research data statistics, etc.

[0036] In another aspect, the present application provides a reagent for simultaneously detecting tumor-specific gene mutation and methylation in one tube, which comprises a capture probe consisting of a probe binding sequence complementary to an adjacent capture probe in the same interval and a target-specific sequence complementary to a tumor-specific mutant gene target interval or a tumor-specific methylated gene target interval.

[0037] Further, the tumor-specific mutant gene is NRAS, CTNNB1, PIK3CA, FBXW7, APC, EGFR, BRAF, CDKN2A, PTEN, FGFR2, HRAS, KRAS, AKT1, TP53, PPP2R1A, GNAS.

[0038] Further, the tumor-specific mutant gene target intervals are chr1:115256462-115256611, chr1:115258679-115258834, chr10:89692723-89693040, chr10:123279607-123279757, chr11:534208-534345, chr12:25378488-25378637, chr12:25380204-25380355, chr12:25398219-25398366, chr14:105246490-105246643, chr17:7572893-7573080, chr17:7573907-7574089, chr17:7576783-7577228, chr17:7577440-7577667, chr17:7578099-7578633, chr17:7579220-7579944, chr19:52715900-52716054, chr20:57484357-57484503, chr3:41266034-41266205, chr3:178916784-178916941, chr3:178921480-178921626, chr3:178936016-178936175, chr3:178951998-178952156, chr4:153247221-153247419, chr4:153249298-153249453, chr4:153251828-153251987, chr5:112175138-112175286, chr5:112175578-112175730, chr7:5571691-5571899, chr7:55232932-55233170, chr7:55241573-55241776, chr7:55242374-55242553, chr7:55248945-55249211, chr7:55259371-55259607, chr7:55268840-55269073, chr7:140453072-140453231, chr9:21971056-21971281.

[0039] Further, the tumor specific methylation genes are SHOX2, PTGER4, H4C6, RASSF1A, Septin9, RNF180, SDC2, PAX1, ZNF671, BMPR1A, PLAC8, EVX1, GNB4, SIX6, PCDHGB7, DAPK1, GSTP1, HIC1, RUNX3, VHL, WT1, BMP3, NDRG4, BCAT1, Twist1, JAM3, TFPI2, CDO1, LYPD5, HOXA9, BNC1, PENK, SFRP1, PCDH8, ADAM23, CHST11, C2orf88, MCF2L, CAMK2N1, TACSTD2.

[0040] Tumor-specific methylation gene target intervals are chr1 :20810296-20810635, chr1 :25255411-25255652, chr1 :59042517-59043026, chr10:88515764-88516078, chr10:88516143-88516235, chr11 :32354800-32355439, chr11 :67350903-67351488, chr11 :133939397-133939638, chr12:25055729-25056569, chr12:104850537-104850822, chr13:53421948-53422217, chr13:53422689-53422828, chr13:113623367-113623449, chr13:113623511-113623603, chr14:60976415-60976602, chr15:83952782-83953090, chr16:58497189-58497988, chr17:1957373-1957754, chr17:75368649-75370546, chr19:44302727-44303241, chr19:58238546-58239068, chr2:191044925-191045141, chr2:207308920-207309240, chr20:21686160-21686478, chr20:21686644-21687729, chr3:10183433-10183998, chr3:50377764-50378580, chr3:157821193-157821570, chr3:179168719-179168984, chr3:179169053-179169633, chr4:81951952-81952848, chr4:84030745-84031349, chr5:40680835-40682071, chr5:63461409-63461789, chr5:63461875-63462146, chr5:115151476-115152553, chr5:140797123-140797741, chr6:26240658-26240991, chr7:19147401-19147925, chr7:27204944-27205261, chr7:27282047-27282879,chr7:27282942-27283176, chr7:93519247-93520492, chr8:41166830-41167127, chr8:41168404-41168548, chr8:57358291-57359351, chr8:97505708-97507647, chr9:90113255-90113681.

[0041] Further, the 5' end of the capture probe is a first probe binding sequence, which is complementary to the 3' end of another capture probe 1, and the 3' end of the capture probe is a second probe binding sequence, which is complementary to the 5' end of another capture probe 2; the length of the first and second probe binding sequences is 8-30 nt, the length of the target-specific sequence is 20-80 nt, and the 5' end of the capture probe is biotin-modified; the other capture probe 1 and the other capture probe 2 are not the same probe.

[0042] Further, the kit further comprises a methylation-sensitive restriction enzyme.

[0043] Further, the kit further comprises exogenous DNA.

[0044] Further, the exogenous DNA is Lambda DNA, which is used to monitor the enzyme cutting efficiency of the methylation-sensitive restriction enzyme.

[0045] Further, the methylation-sensitive restriction enzyme is selected from HpaI, HpaII, HhaI, AciI, and combinations thereof.

[0046] In another aspect, the present application provides the use of the above-mentioned reagent in the preparation of a tumor mutation and methylation condition diagnosis reagent.

[0047] Compared with the prior art, the beneficial effects of the present application include:

[0048] (1) The present application can accurately detect the point mutation and insertion-deletion variation, methylation state of 55 genes (including cancer driver genes, tumor suppressor genes) at the same time. In the prior art, the mutation and methylation state are often detected separately, and the problem of simultaneously detecting multiple cancer gene mutations and methylation states has not been solved. The present application can simultaneously detect multiple cancer gene mutations and methylation states in one tube.

[0049] (2) Based on the tumor-related gene hotspot designed above, the application carries out targeted detection, including point mutations and insertion and deletion variations of tumor-specific genes related to multiple cancers such as lung cancer, colorectal cancer, esophageal cancer, liver cancer, gastric cancer, bladder cancer, cervical cancer, breast cancer, ovarian cancer, and methylation state, so that multiple cancer gene mutation and methylation state information can be obtained through one detection, thereby giving guidance suggestions for early detection of tumors.

[0050] (3) The enzyme cutting efficiency of the application for free DNA is greater than 99%, the enzyme cutting recovery rate is greater than 95%; the ligation enzyme cutting recovery rate is greater than 50%, which is similar to the simple ligation recovery rate, and has no effect on the number of mutation fragments of related genes.

[0051] (4) The hybridization capture time of the application only needs 2h, and at most 10 samples can be hybridized at one time, thereby saving cost and time.

[0052] (5) The application can obtain high-quality high-throughput sequencing data (Q30>90%), and only 0.5G of data amount is needed for each sample.

[0053] (6) The alignment rate of the probe of the application can reach 98% (the proportion of data alignment to the genome), the capture efficiency (the ratio of the number of target region sequences to the total sequence determined) is greater than 40%, and the uniformity (0.2x average coverage percentage) is greater than 90%. The coverage, capture efficiency, and uniformity are directly related to the design of the target. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 is a schematic diagram of the methylation and mutation targeted detection experiment process and operation time;

[0055] Figure 2 is a schematic diagram of the ligation enzyme cutting recovery result of free DNA in Example 2;

[0056] Figure 3 is a schematic diagram of the quality effect of the sample in Examples 3, 4, 5, and 6;

[0057] Figure 4 is a schematic diagram of the variation detection result of the mutation standard in Example 3;

[0058] Figure 5 is a schematic diagram of the methylation detection result of the methylation standard in Example 4;

[0059] Figure 6 is a schematic diagram of the partial methylation site detection result of the lung cancer sample in Example 5;

[0060] Figure 7 is a schematic diagram of the partial methylation site detection result of the liver cancer sample in Example 6. DETAILED DESCRIPTION

[0061] The basic operation steps are shown in Figure 1.

[0062] Detection steps:

[0063] 1. Select the methylation and mutation sites of cancer-related genes in Table 1 and their upstream and downstream extensions of 30-100 bp as target intervals by database.

[0064] 2. Synthesize capture probes corresponding to the target interval, which include probe binding sequences complementary to another probe and target-specific sequences complementary to the nucleic acid target sequence, the length of the probe binding sequence is 8-30 nt, the length of the target-specific sequence is 20-80 nt, and the 5' end is biotin-modified.

[0065] 3. Sample DNA extraction and mixing with Lambda DNA

[0066] Use the plasma free DNA or exfoliated cell DNA extraction kit to extract the sample DNA, and the method refers to the extraction kit steps. The genomic DNA sample needs to be fragmented into 180-220 bp DNA fragments, and the free DNA is directly mixed with a certain proportion of Lambda DNA (when the sample input is 10 ng≤n<40 ng, Lambda DNA is added 0.165 pg; when the sample input is <10 ng, Lambda DNA is added 0.05 pg).

[0067] 4. End repair, "A" addition and ligation by T4 DNA ligase, etc. using End Repair & A-Tailing Enzyme to connect the universal adapter to the DNA fragments (related reagents from NadPrep DNA Library Preparation Kit (for Illumina) E96), and obtain 17-34 μL of sample ligation product after purification.

[0068] 5. Enzymatic digestion library construction by methylation-sensitive restriction enzyme HhaI

[0069] Use methylation-sensitive restriction enzymes to process sample ligation products, connect index adapters to DNA fragments by PCR amplification, and obtain sample DNA methylation and mutation pre-library after purification;

[0070] 5.1 Configure the MSRE reaction solution according to the following table, vortex mix the prepared reaction system, short centrifuge, and place on ice for standby.

[0071] Note: All reagents should be melted and thoroughly mixed before use. The melted reagents are placed in an ice box for standby, and should be immediately returned to the storage condition after use.

[0072] 5.2 Add 3 μL of MSRE reaction to the sample tube from step 4, vortex to mix, spin briefly, and run the following program on a PCR machine (set the hot lid temperature to 90°C):

[0073] 5.3 Purification after MSRE reaction

[0074] After the MSRE reaction, use the purification magnetic beads AMPure beads for purification. Note that the purification magnetic beads AMPure beads should be equilibrated at room temperature for at least 30 min before use;

[0075] a. Equilibrate the purification magnetic beads AMPure beads at room temperature for at least 30 min;

[0076] b. Add 30 μL of Nuclease Free Water to the 20 μL sample, and vortex to mix;

[0077] c. Add 90 μL of AMPure beads to the reaction system, and vortex to mix evenly;

[0078] d. Incubate the mixture at room temperature for 5-10 min, then place it on a magnetic stand, and let it stand for about 3-5 min until the magnetic beads are completely separated from the solution, then carefully remove the supernatant.

[0079] e. Wash the magnetic beads with 200 μL of 80% ethanol. Repeat once, for a total of 2 washes.

[0080] f. Air-dry the magnetic beads on the magnetic stand until they are dry. [Note: Do not over-dry or wet the magnetic beads]

[0081] g. Resuspend the dried magnetic beads with 20 μL of Nuclease Free Water, and incubate at room temperature for about 5 min. Place the centrifuge tube on the magnetic stand, and when the supernatant is clear, transfer the supernatant to a new PCR tube and place it on ice for standby.

[0082] 5.4 PCR amplification to connect the DNA fragments with index adapters

[0083] a. Configure the amplification reaction solution according to the following table, vortex to mix the prepared reaction system, spin briefly, and place it on ice for standby.

[0084] Note: All reagents should be melted and thoroughly mixed before use. Place the melted reagents in an ice box for standby, and please return them to the storage condition immediately after use.

[0085] b. Add 5 μL of Universal UDI-Index Primer Mix, make sure to record the index corresponding to each sample, then add 25 μL of amplification reaction solution to the reaction tube, vortex mix, centrifuge briefly, and run the following program on the PCR instrument (heat cover setting: 105℃):

[0086] 5.5 Purification of pre-library

[0087] After the amplification reaction, purification is performed according to the following operation. Purification is performed using AMPure beads. Note that the AMPure beads should be equilibrated at room temperature for at least 30 min before use.

[0088] a. Add 50 μL of AMPure beads to the PCR tube containing the amplification reaction product from step 5.4.b and mix well.

[0089] b. Follow the instructions in 5.4.d-g.

[0090] 6. Quality control of pre-library

[0091] The pre-library is subjected to concentration quality control using the HS dsDNA reagent and Qubit 4.0 instrument. To meet the requirements of the downstream pooling, the Qubit concentration should be ≥ 30 ng / μL.

[0092] 7. Pooling of pre-library

[0093] During pooling, 10 samples are mixed according to the rule of 500 ng / sample.

[0094] 8. Concentration of pre-library after pooling.

[0095] 8.1 Add 1.8 times the volume of AMPure beads to the total volume of the library after pooling, and vortex mix.

[0096] 8.2 Follow the instructions in 5.4.d-f.

[0097] 8.3 Add 19 μL of Nuclease Free Water to the dried centrifuge tube, vortex mix, and incubate at room temperature for 5 min.

[0098] 8.4 Centrifuge the centrifuge tube briefly and place it on the magnetic stand for 2-5 min until the liquid is completely clear. Use a pipette to transfer all the supernatant to a new 0.2 mL PCR tube.

[0099] 9. Library hybridization

[0100] 9.1 System configuration

[0101] Prepare the library hybridization reaction solution according to the following table:

[0102] Note: All reagents should be thawed and mixed well before use. Thawed reagents should be placed on ice until use. Please return the reagents to the storage condition immediately after use.

[0103] Vortex the prepared reaction system, centrifuge briefly, and place on ice until use.

[0104] 9.2 Library hybridization reaction

[0105] Add 40 μL of the hybridization reaction solution to the product of 8.4, and separately add 2 μL of Custom panel probe, vortex well, and briefly centrifuge. Run the following program on the PCR instrument (set the hot lid to 105°C):

[0106] 10. Library capture and elution

[0107] 10.1 Magnetic bead cleaning

[0108] a. Vortex the Streptavidin Beads that have been equilibrated at room temperature for at least 30 min for 15 sec to ensure complete mixing;

[0109] b. Mix and clean n x 25 μL Streptavidin Beads in a 0.2 mL centrifuge tube (n is the total number of capture reactions, and n ≤ 5; when n > 5, multiple tubes should be used for mixing and cleaning).

[0110] c. Place the Streptavidin Beads on the magnetic stand for about 2 min until the liquid is completely clarified, and discard the supernatant using a pipette.

[0111] d. Remove the centrifuge tube from the magnetic stand, add 150 μL of preheated Wash Buffer A Pro, and gently blow and mix for more than 10 times.

[0112] e. Place the centrifuge tube on the magnetic stand for about 2 min until the liquid is completely clarified, and discard the supernatant using a pipette.

[0113] f. Repeat steps d and e once (Note: Wash Buffer A Pro should be heated back to 50-60°C).

[0114] g. After centrifuging the centrifuge tube, place it on the magnetic stand for 10 sec, and use a 10 μL pipette to discard all the Wash Buffer A Pro at the bottom of the tube.

[0115] h. Take n x 9 μL of Hyb#1 resuspended Streptavidin Beads, gently pipette and mix for more than 10 times.

[0116] 10.2 Magnetic bead capture and elution

[0117] a. After the hybridization reaction, proceed to the capture step, keep the PCR machine running.

[0118] b. Keep the PCR tubes in the PCR machine, take 8 μL of resuspended Streptavidin Beads and immediately add to each hybridization reaction, gently pipette and mix for more than 10 times.

[0119] c. Incubate at 60°C for 10 min.

[0120] d. After incubation, remove the PCR tubes from the PCR machine and place them on the magnetic stand for 2 min, until the liquid is completely clarified, use a pipette to discard the supernatant (try to remove the supernatant as completely as possible).

[0121] e. Remove the PCR tubes from the magnetic stand, add 150 μL of preheated Wash Buffer A Pro, gently pipette and mix for more than 10 times (avoid air bubbles).

[0122] f. Place the PCR tubes on the magnetic stand for 2 min, until the liquid is completely clarified, use a pipette to discard the supernatant (try to remove the supernatant as completely as possible).

[0123] g. Remove the PCR tubes from the magnetic stand, add 100 μL of preheated Wash Buffer A Pro, gently pipette and mix for more than 10 times, transfer the reaction to a new PCR tube (note: try to avoid air bubbles in this step, air bubbles touching the tube cap can cause a decrease in target capture rate).

[0124] h. Place the PCR tubes in the PCR machine, incubate at 60°C for 3 min.

[0125] i. After incubation, remove the PCR tubes from the PCR machine and place them on the magnetic stand for 2 min, until the liquid is completely clarified, use a pipette to discard the supernatant (try to remove the supernatant as completely as possible).

[0126] j. Add 150 μL of room temperature Wash Buffer B to the PCR tubes, gently pipette and mix for more than 10 times.

[0127] k. Place the PCR tubes on the magnetic stand for 2 min, until the liquid is completely clarified, use a pipette to discard the supernatant.

[0128] l. After centrifuging the PCR tubes, place them on the magnetic stand, use a 10 μL pipette tip to remove a small amount of residual liquid, be careful not to aspirate the magnetic beads.

[0129] m. Add 22.5 μL Nuclease Free Water, resuspend the beads by pipetting up and down 10 times and transfer the resuspended beads to a new PCR tube.

[0130] 11. Hybrid Capture Library PCR Amplification Purification

[0131] 11.1 Hybrid Capture Library PCR Reaction Configuration

[0132] Configure the Hybrid Capture Library PCR reaction solution according to the following table:

[0133] Note: All reagents should be melted and thoroughly mixed before use. The melted reagents should be placed in an ice box for use. Please return them to the storage condition immediately after use.

[0134] Vortex the configured reaction system, centrifuge briefly, and place it on ice for use.

[0135] 11.2 Hybrid Capture Library PCR Reaction

[0136] Add the Hybrid Capture Library PCR reaction solution to the product of 11.1.m, vortex to mix, and run the following program on the PCR instrument (heat cover setting: 105°C):

[0137] After the Hybrid Capture Library PCR reaction, use the purification magnetic beads AMPure beads to purify the PCR amplification product to obtain the captured sample library.

[0138] 12. Hybrid Capture Library Quality Control

[0139] 12.1 Concentration Quality Control

[0140] Use the HS dsDNA reagent and Qubit 4.0 instrument to perform concentration quality control on the Hybrid Capture Library. To meet the requirements of downstream sequencing, the concentration of the Hybrid Capture Library should be ≥1.5 ng / μL.

[0141] 12.2 Fragment Quality Control

[0142] Use Qsep-1 / Labchip GX Touch (Caliper) to determine the library fragment size. The average length of the fragments should be distributed in the range of 200-1000 bp.

[0143] 13. Sequencing

[0144] The application uses a second-generation sequencing platform familiar to those skilled in the art to perform high-throughput sequencing, and the captured sequences are subjected to sequence determination on an Illumina sequencing platform using a method of sequencing by synthesis.

[0145] 14. Data processing and analysis

[0146] 14.1 Variants data is analyzed, and the results are filtered using fastp. The filtering conditions are as follows: variants meeting the following filtering conditions are retained: base quality ≥ 20, sequence alignment quality ≥ 5, and strand bias < 90%.

[0147] 14.2 bwa alignment to the reference genome.

[0148] 14.3 Filtering of unmapped reads, secondary alignment or multiple alignment reads.

[0149] 14.4 Removal of PCR duplicates, analysis of methylation and mutation levels.

[0150] The application will be further described below in conjunction with specific examples.

[0151] Example 1 Screening of methylation and mutation sites related to human tumors and probe design method

[0152] In this study, first, the GEO was used to select pan-cancer (here, pan-cancer is defined as all cancer types in the TCGA pan-cancer data, including urothelial carcinoma, breast cancer, cervical cancer, cholangiocarcinoma, colon cancer, esophageal cancer, hepatocellular carcinoma, lung adenocarcinoma, lung squamous carcinoma, pancreatic cancer, prostate cancer, rectal cancer, gastric cancer, endometrial cancer, etc.) sample data, to screen for aberrant methylation-differentially expressed genes in tumors, perform functional enrichment analysis using Metascape, screen for important module genes using Cytoscape, then use the UALCAN database to verify the aberrant methylation-differentially expressed genes, further use the R language to screen for methylation target genes affecting the prognosis of specific cancers using TCGA cancer data, and perform survival and correlation analysis of methylation CpG sites. Finally, GSEA enrichment analysis was performed on the screened key methylation target genes, and correlation analysis of the methylation target genes and clinical pathological characteristics was performed. In combination with the methylation gene sites screened from the literature and TCGA data, the methylation detection panel was finally combined.

[0153] Based on the articles published in recent years in the field of cancer mutation spectrum and screening and the sequencing data in COSMIC database, the mutation genes and mutation sites with higher correlation with cancer occurrence were screened out. The keywords such as liquid biopsy, ctDNA, somatic mutation, tumor screening, cancer, early cancer screening were used for literature search in PubMed database. According to the search results, the mutation genes and sites related to cancer were sorted out. In COSMIC database, Cancer Browser was selected, and then in Tissue selection, Histology selection, Sub-histology selection, a certain cancer was selected, and the sequencing data of all samples of the cancer in the database were sorted out. The mutation sites with more than 5 samples were selected from the top 20 genes in mutation frequency. The genes and sites screened out in combination with literature and COSMIC data were finally combined into a mutation detection panel.

[0154] The methylation detection panel and the mutation detection panel were combined together to obtain the panel interval for co-detection of methylation and mutation as shown in Table 1.

[0155] Table 1: Selected cancer-related gene capture region

[0156] Note: Chr: chromosome; Start: interval start position; End: interval end position.

[0157] Capture probes were designed for the interval in Table 1. The probe sequence included a probe binding sequence complementary to another probe and a target-specific sequence complementary to the nucleic acid target sequence. The length of the probe binding sequence was 8-30 nt, and the length of the target-specific sequence was 20-80 nt. The 5' end was biotinylated. Part of the probe sequence is shown in Table 2.

[0158] Table 2: Part of the probe sequence example

[0159] Example 2: Effect of methylation restriction enzyme on mutation fragment detection

[0160] The sample in this example was 1 plasma sample of a healthy person, and the effect of methylation restriction enzyme on mutation fragment detection was evaluated, and the specific operation was as follows:

[0161] (1) The cfDNA was extracted according to the method described above, and the concentration was determined to be 0.91 ng / μL using Qubit 4.0 after extraction. 5 μL of cfDNA sample was used for real-time fluorescent quantitative PCR detection of actin gene, and recorded as CT1.

[0162] (2) Take 10 ng, i.e. 10.99 μL of cfDNA for end repair plus A, adapter ligation, and purification after adapter ligation. After purification of the ligation, 35 μL of nuclease-free water is used for elution, and 5 μL of the eluate is used for real-time fluorescent quantitative PCR detection of the actin gene, which is recorded as CT2;

[0163] (3) Take 17 μL of (2) for MSRE reaction; after MSRE reaction and purification of the MSRE reaction, 20 μL of nuclease-free water is used for elution, and 5 μL of the eluate is used for real-time fluorescent quantitative PCR detection of the actin gene, which is recorded as CT3;

[0164] (4) After completion of the real-time fluorescent quantitative PCR detection of the actin gene, the connection recovery efficiency, the enzyme cutting recovery efficiency, and the connection enzyme cutting recovery efficiency are calculated according to the difference between the CT values, and the specific calculation formula is as follows:

[0165] a) Connection recovery efficiency = 1 / 2^(CT2-CT1)*35 / 10.99

[0166] b) Enzyme cutting recovery efficiency = 1 / 2^(CT3-CT2)*17 / 20

[0167] c) Connection enzyme cutting recovery efficiency = connection recovery efficiency*enzyme cutting recovery efficiency

[0168] The detection results are shown in Table 3 and FIG. 2. The results show that the enzyme cutting efficiency of the mutation / methylation co-detection method for free DNA is greater than 99%, the enzyme cutting recovery rate is greater than 95%; the connection enzyme cutting recovery rate is greater than 50%, which is similar to the simple connection recovery rate, and has no effect on the number of mutant fragments of related genes.

[0169] Table 3 Detection results of Example 2

[0170] The quality of the samples in the following Examples 3, 4, 5, and 6 is shown in FIG. 3.

[0171] Accuracy of gene mutation detection in Example 3

[0172] The sample in this example is a gDNA mutation standard product of Horizon Discovery Company, with product catalog number HD827. The standard product contains multiple mutation sites verified by digital PCR.

[0173] Firstly, the gDNA mutation standard was physically broken into 200-300 bp fragments, and then 40 ng was put in, and the end repair plus A, adapter ligation, MSRE reaction, sub-library enrichment, sub-library pooling, hybrid capture of target sequences, cleaning and enrichment purification, library quality control, machine sequencing and data processing analysis were completed according to the previous steps 3-14.

[0174] According to the sequencing results, the mutation frequency of the mutation site was obtained. The detection results are shown in Table 4 and Figure 4. The results show that the mutation frequency of the mutation site obtained by using the mutation / methylation co-detection method to detect the mutation standard is basically close to the theoretical value of the standard. It can be seen that this method has high accuracy for the detection of related gene mutations.

[0175] Table 4 Detection results of Example 3 Note: Cosmic ID represents the gene written in the Cosmic database, Ref is the normal type, Alt is the type after gene mutation, DEL represents deletion, and SNP represents single base mutation.

[0176] Accuracy of methylation detection in Example 4

[0177] The sample of this example is the gDNA negative methylation standard and positive methylation standard of Zymo Research Company, and the methylation sites in the standard are verified by NGS.

[0178] Firstly, the gDNA methylation standard was physically broken into 200-300 bp fragments, and then 40 ng was put in, and the end repair plus A, adapter ligation, MSRE reaction, sub-library enrichment, sub-library pooling, hybrid capture of target sequences, cleaning and enrichment purification, library quality control, machine sequencing and data processing analysis were completed according to the previous steps 3-14.

[0179] According to the sequencing results, the methylation level was obtained. The detection results are shown in Table 5 and Figure 5. The results show that the detection value obtained by using the mutation / methylation co-detection method to detect the methylation standard is basically close to the theoretical value. It can be seen that this method has high accuracy for the detection of related gene methylation.

[0180] Table 5

[0181] Example 5 Detection of actual lung cancer samples

[0182] The sample of this example is derived from a right upper lung infiltrating adenocarcinoma IV stage lung cancer patient, and the sample source is the plasma separated from the peripheral blood of the lung cancer patient before surgery.

[0183] Firstly, free DNA of the plasma sample was extracted, and the concentration of the extracted free DNA was 0.7 ng / μL, then 26 ng was taken and put in, and the end repair plus A, adapter ligation, MSRE reaction, sub-library enrichment, sub-library pooling, hybridization capture of target sequences, cleaning and enrichment purification, library quality inspection, machine sequencing, and data processing analysis were completed according to the foregoing steps 3-14.

[0184] According to the sequencing results, the methylation and mutation results were obtained. The detection results are shown in Table 6 and Figure 6. As shown in Table 6, the KRAS gene of the sample has G35T variation, and the mutation frequency is 2.91%. As shown in Figure 5, the lung cancer related methylation site level of the sample is significantly different from that of a normal person.

[0185] Table 6 actual sample detection 1 results

[0186] Example 6 actual liver cancer sample detection

[0187] The sample of this example is derived from a liver cancer patient in stage IV hepatocellular carcinoma, and the sample source is the plasma separated from the peripheral blood of the liver cancer patient before surgery.

[0188] Firstly, free DNA of the plasma sample was extracted, and the concentration of the extracted free DNA was 3.0 ng / μL, then 40 ng was taken and put in, and the end repair plus A, adapter ligation, MSRE reaction, sub-library enrichment, sub-library pooling, hybridization capture of target sequences, cleaning and enrichment purification, library quality inspection, machine sequencing, and data processing analysis were completed according to the foregoing steps 3-14.

[0189] According to the sequencing results, the methylation and mutation results were obtained. The detection results are shown in Table 7 and Figure 7. As shown in Table 7, the TP53 gene of the sample has G422A variation, and the mutation frequency is 1.69%. As shown in Figure 6, the liver cancer related methylation site level of the sample is significantly different from that of a normal person.

[0190] Table 7 actual sample detection 2 results

[0191] The above results show that the detection method provided in the present application has high sensitivity and wide coverage. One can simultaneously detect the methylation and mutation of 55 tumor related genes. The embodiments described above are only a few embodiments of the present application, and the description is more specific, but it cannot be understood as limiting the patent scope of the present application. Therefore, the protection scope of the patent of the present application is subject to the appended claims.

Claims

1. A method for simultaneously detecting tumor-specific gene mutations and methylation in a tube, comprising, The method comprises the following steps: (1) selecting tumor-specific mutant genes and tumor-specific methylation genes, selecting mutation sites of tumor-specific mutant genes and methylation sites of tumor-specific methylation genes, and selecting the mutation sites of tumor-specific mutant genes and the methylation sites of tumor-specific methylation genes and the regions of 30-100 bp extended therefrom as tumor-specific mutant gene target intervals and tumor-specific methylation gene target intervals; (2) synthesizing target interval corresponding paired capture probes, which are composed of probe combination sequences complementary to adjacent capture probes in the same interval and target-specific sequences complementary to tumor-specific mutant gene target intervals or tumor-specific methylation gene target intervals; (3) extracting free DNA or genomic DNA from a sample to be tested, mixing with exogenous DNA, end-repairing, adding "A", and ligating, and obtaining sample ligation products of the sample after purification; (4) treating the ligation products of the sample with methylation-sensitive restriction endonucleases, amplifying the DNA fragments by PCR to connect index adapters, and obtaining sample DNA methylation and mutation pre-library after purification; (5) hybridizing the DNA methylation and mutation pre-library in step (4) with the capture probes to capture the methylation and mutation DNA sequences in the target region; (6) PCR amplifying the DNA sequences in the target region to obtain amplification products; (7) purifying the amplification products in step (6), performing quality inspection, and obtaining the methylation and mutation library of the target region; (8) performing high-throughput sequencing on the methylation and mutation library of the target region constructed in step (7) to obtain the sequence of the target fragment; (9) comparing the sequence information of the target fragment obtained with normal gene sequences to obtain mutation and methylation information of the target fragment; The method is used for non-diagnostic purposes.

2. The method of claim 1, wherein in step (1), the methylation and mutation sites of cancer-related genes are selected in combination with literature and TCGA and COSMIC data.

3. The method of claim 1, wherein the tumor-specific mutant gene is NRAS, CTNNB1, PIK3CA, FBXW7, APC, EGFR, BRAF, CDKN2A, PTEN, FGFR2, HRAS, KRAS, AKT1, TP53, PPP2R1A, or GNAS.

4. The method of claim 3, the tumor-specific mutant gene target intervals are chrl: 115256462-115256611, chrl: 115258679-115258834, chr 10: 89692723-89693040, chr 10: 123279607-123279757, chrll: 534208-534345, chr 12: 25378488-25378637, chr 12: 25380204-25380355, chr 12: 25398219-25398366, chr 14: 105246490-105246643, chr 17: 7572893-7573080, chr 17: 7573907-7574089, chr 17: 7576783-7577228, chr 17: 7577440-7577667, chr 17: 7578099-7578633, chr 17: 7579220-7579944, chr 19: 52715900-52716054, chr20: 57484357-57484503, chr3: 41266034-41266205, chr3: 178916784-178916941, chr3: 178921480-178921626, chr3: 178936016-178936175, chr3: 178951998-178952156, chr4: 153247221-153247419, chr4: 153249298-153249453, chr4: 153251828-153251987, chr5: 112175138-112175286, chr5: 112175578-112175730, chr7: 5571691-5571899, chr7: 55232932-55233170, chr7: 55241573-55241776, chr7: 55242374-55242553, chr7: 55248945-55249211, chr7: 55259371-55259607, chr7: 55268840-55269073, chr7: 140453072-140453231, chr9: 21971056-21971281.

5. The method of claim 1, wherein the tumor-specific methylation gene is SHOX2, PTGER4, H4C6, RASSF1A, Septin9, RNF180, SDC2, PAX1, ZNF671, BMPR1A, PLAC8, EVX1, GNB4, SIX6, PCDHGB7, DAPK1, GSTP1, HIC1, RUNX3, VHL, WT1, BMP3, NDRG4, BCAT1, Twist1, JAM3, TFPI2, CDO1, LYPD5, HOXA9, BNC1, PENK, SFRP1, PCDH8, ADAM23, CHST11, C2orf88, MCF2L, CAMK2N1, TACSTD2.

6. The method of claim 5, wherein the tumor-specific methylation gene target intervals are chr1 :20810296-20810635, chr1 :25255411-25255652, chr1 :59042517-59043026, chr10:88515764-88516078, chr10:88516143-88516235, chr11 :32354800-32355439, chr11 :67350903-67351488, chr11 :133939397-133939638, chr12:25055729-25056569, chr12:104850537-104850822, chr13:53421948-53422217, chr13:53422689-53422828, chr13:113623367-113623449, chr13:113623511-113623603, chr14:60976415-60976602, chr15:83952782-83953090, chr16:58497189-58497988, chr17:1957373-1957754, chr17:75368649-75370546, chr19:44302727-44303241, chr19:58238546-58239068, chr2:191044925-191045141, chr2:207308920-207309240, chr20:21686160-21686478, chr20:21686644-21687729, chr3:10183433-10183998, chr3:50377764-50378580, chr3:157821193-157821570, chr3:179168719-179168984, chr3:179169053-179169633, chr4:81951952-81952848, chr4:84030745-84031349, chr5:40680835-40682071, chr5:63461409-63461789, chr5:63461875-63462146, chr5:115151476-115152553, chr5:140797123-140797741, chr6:26240658-26240991, chr7:19147401-19147925, chr7:27204944-27205261,chr7:27282047-27282879, chr7:27282942-27283176, chr7:93519247-93520492, chr8:41166830-41167127, chr8:41168404-41168548, chr8:57358291-57359351, chr8:97505708-97507647, chr9:90113255-90113681.

7. The method of claim 1, wherein the mutation site includes point mutation and indel mutation.

8. The method of claim 1, wherein the methylation site includes a site recognized by methylation-sensitive restriction enzyme.

9. The method of claim 1, wherein the methylation-sensitive restriction enzyme is selected from the group consisting of HpaI, HpaII, HhaI, AciI and combinations thereof.

10. The method of claim 1, wherein the 5' end of the capture probe comprises a first probe binding sequence complementary to the 3' end of another capture probe 1, and the 3' end of the capture probe comprises a second probe binding sequence complementary to the 5' end of another capture probe 2; the first and second probe binding sequences have a length of 8-30 nt, the target-specific sequence has a length of 20-80 nt, the 5' end of the capture probe is biotin-modified; and the another capture probe 1 and the another capture probe 2 are not the same probe.

11. The method of claim 1, wherein the genomic DNA is extracted from a cell line, tumor tissue or exfoliated cells, or the cell-free DNA is extracted from plasma, urine, pleural effusion or ascites in step (3).

12. The method of claim 1, wherein the sample ligation product in step (3) comprises all sequences of the sample DNA and the added exogenous DNA.

13. The method of claim 11, wherein the exogenous DNA is Lambda DNA, which is used to monitor the cleavage efficiency of the methylation-sensitive restriction enzyme.

14. The method of claim 1, wherein 1-10 sample pre-libraries are hybridized simultaneously in step (5).

15. The method of claim 1, wherein the library concentration is greater than or equal to 1.5 ng / μL, and the average fragment size is distributed in 200-1000 bp.

16. A kit for simultaneous detection of tumor-specific gene mutation and methylation, comprising: a) a primer set for detecting a tumor-specific gene mutation; b) a primer set for detecting a tumor-specific gene methylation; and c) a primer set for detecting a reference gene mutation and methylation. The reagents comprise capture probes, which comprise a probe binding sequence complementary to an adjacent capture probe in the same interval and a target-specific sequence complementary to a target interval of a tumor-specific mutation gene or a target interval of a tumor-specific methylation gene.

17. The agent of claim 16, the tumor-specific mutant gene is NRAS, CTNNB1, PIK3CA, FBXW7, APC, EGFR, BRAF, CDKN2A, PTEN, FGFR2, HRAS, KRAS, AKT1, TP53, PPP2R1A, GNAS.

18. The agent of claim 17, the tumor-specific mutant gene target interval is chrl: 115256462-115256611, chrl: 115258679-115258834, chr10: 89692723-89693040, chr10: 123279607- 123279757, chrll: 534208-534345, chr12: 25378488-25378637, chr12: 25380204-25380355, chr12: 25398219-25398366, chr14: 105246490-105246643, chr17: 7572893-7573080, chr17: 7573907-7574089, chr17: 7576783-7577228, chr17: 7577440-7577667, chr17: 7578099-7578633, chr17: 7579220-7579944, chr19: 52715900-52716054, chr20: 57484357-57484503, chr3: 41266034-41266205, chr3: 178916784-178916941, chr3: 178921480-178921626, chr3: 178936016-178936175, chr3: 178951998-178952156, chr4: 153247221-153247419, chr4: 153249298-153249453, chr4: 153251828-153251987, chr5: 112175138-112175286, chr5: 112175578-112175730, chr7: 5571691-5571899, chr7: 55232932-55233170, chr7: 55241573-55241776, chr7: 55242374-55242553, chr7: 55248945-55249211, chr7: 55259371-55259607, chr7: 55268840-55269073, chr7: 140453072-140453231, chr9: 21971056-21971281.

19. The reagent of claim 16, wherein the tumor-specific methylation gene is SHOX2, PTGER4, H4C6, RASSF1A, Septin9, RNF180, SDC2, PAX1, ZNF671, BMPR1A, PLAC8, EVX1, GNB4, SIX6, PCDHGB7, DAPK1, GSTP1, HIC1, RUNX3, VHL, WT1, BMP3, NDRG4, BCAT1, Twist1, JAM3, TFPI2, CDO1, LYPD5, HOXA9, BNC1, PENK, SFRP1, PCDH8, ADAM23, CHST11, C2orf88, MCF2L, CAMK2N1, TACSTD2.

20. The reagent of claim 19, wherein the tumor-specific methylated gene target interval is chrl:20810296-20810635, chrl:25255411-25255652, chrl:59042517-59043026, chr10:88515764-88516078, chr10:88516143-88516235, chrll:32354800-32355439, chrll:67350903-67351488, chrll:133939397-133939638, chr12:25055729-25056569, chr12:104850537-104850822, chr13:53421948-53422217, chr13:53422689-53422828, chr13:113623367-113623449, chr13:113623511-113623603, chr14:60976415-60976602, chr15:83952782-83953090, chr16:58497189-58497988, chr17:1957373-1957754, chr17:75368649-75370546, chr19:44302727-44303241, chr19:58238546-58239068, chr2:191044925-191045141, chr2:207308920-207309240, chr20:21686160-21686478, chr20:21686644-21687729, chr3:10183433-10183998, chr3:50377764-50378580, chr3:157821193-157821570, chr3:179168719-179168984, chr3:179169053-179169633, chr4:81951952-81952848, chr4:84030745-84031349, chr5:40680835-40682071, chr5:63461409-63461789, chr5:63461875-63462146, chr5:115151476-115152553, chr5:140797123-140797741, chr6:26240658-26240991, chr7:19147401-19147925, chr7:27204944-27205261,chr7:27282047-27282879, chr7:27282942-27283176, chr7:93519247-93520492, chr8:41166830-41167127, chr8:41168404-41168548, chr8:57358291-57359351, chr8:97505708-97507647, chr9:90113255-90113681.

21. The reagent of claim 16, wherein the 5' end of the capture probe is a first probe binding sequence, the first probe binding sequence is complementary to the 3' end of another capture probe 1, and the 3' end of the capture probe is a second probe binding sequence, the second probe binding sequence is complementary to the 5' end of another capture probe 2; the length of the first and second probe binding sequences is 8-30 nt, the length of the target-specific sequence is 20-80 nt, the 5' end of the capture probe is biotin-modified; the other capture probe 1 and the other capture probe 2 are not the same probe.

22. The reagent of claim 16, further comprising a methylation-sensitive restriction enzyme.

23. The reagent of claim 22, wherein the methylation-sensitive restriction enzyme is selected from the group consisting of HpaI, HpaII, HhaI, AciI, and combinations thereof.

24. Use of the reagent of any one of claims 16-23 in the preparation of a tumor mutation and methylation status diagnostic reagent.

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