CN114214408A

CN114214408A - Method, probe library and kit for detecting tumor ctDNA methylation with high throughput and high sensitivity

Info

Publication number: CN114214408A
Application number: CN202111579790.XA
Authority: CN
Inventors: 唐超; 施小龙; 吴永忠; 王颖; 綦俊
Original assignee: Chongqing University Cancer Hospital
Current assignee: Chongqing University Cancer Hospital
Priority date: 2021-12-22
Filing date: 2021-12-22
Publication date: 2022-03-22
Anticipated expiration: 2041-12-22
Also published as: CN114214408B

Abstract

The invention relates to the technical field of biology, and particularly discloses a method, a probe library and a kit for detecting tumor ctDNA methylation with high flux and high sensitivity. The method has the advantages of high sensitivity, high flux and digital analysis, and the method does not need to carry out bisulfite conversion, thereby effectively avoiding the problems of fragmentation and incomplete conversion of the ctDNA treated by the bisulfite.

Description

Method, probe library and kit for detecting tumor ctDNA methylation with high throughput and high sensitivity

Technical Field

The invention relates to the technical field of biology, in particular to a method for detecting DNA methylation with high flux and high sensitivity.

Background

Cancer is a general term for a series of malignant tumors and is one of the leading causes of human death worldwide. In 2020, 996 ten thousand cases of cancer deaths worldwide, of which 180 ten thousand lung cancer deaths account for 18% of the total number of cancer deaths, being the highest cancer mortality worldwide. The main treatment methods of cancer include surgical treatment, radiotherapy, chemotherapy, molecular targeted therapy and the like, but the average five-year survival rate of patients with advanced cancer is only 26%. However, cancer patients who are discovered, diagnosed and treated early often have better treatment effect, and the average five-year survival rate is 91%, so early tumor screening can effectively reduce the death rate.

Early screening of tumors has been mainly performed by conventional detection and liquid biopsy. Traditional tests include tumor markers, endoscopy, imaging, and the like. The number of tumor markers is large, and false positive is high; the time of finding the tumor focus by imaging is too late; endoscopy is only suitable for some cancer types such as gastric cancer, colorectal cancer and the like, and the adaptability of patients is low. In recent years, the role of liquid biopsy in cancer screening and treatment has rapidly been gaining importance. The liquid biopsy can obtain information of base mutation, methylation and copy number variation by detecting Circulating Tumor DNA (ctDNA) in blood, and has the advantages of simple sampling, high sensitivity and high specificity.

ctDNA can be used for early screening and process detection of various tumors (such as breast cancer, ovarian cancer, leukemia and lung cancer) by means of detecting methylation, gene mutation and the like. ctDNA methylation is considered as one of the main means for early diagnosis of cancer, and early detection of ctDNA methylation can detect cancer four years earlier than conventional blood diagnosis. In contrast to the high degree of individualization of gene mutations, methylation detection generally targets the promoter region of a particular gene. The early stage of tumorigenesis is usually accompanied by abnormal promoter methylation, the methylation is a common characteristic of different cancers, and ctDNA accurately distinguishes 50 kinds of cancers (colorectal cancer, esophageal cancer, liver cancer, lung cancer, ovarian cancer and the like) in different stages by using methylation information. Abnormal DNA methylation can be used as a biomarker, and studies have shown that STRATIFIN gene is methylated in 96% of breast cancer patients and GSTP1 gene is methylated in more than 90% of prostate cancer patients. The ctDNA methylation detection biological sample is easy to obtain, when tumors occur, the tumor cell DNA is released into peripheral blood, is enriched in serum and plasma, has the DNA content which is several times higher than that of the peripheral blood of normal people, and can detect the promoter methylation in the peripheral blood plasma, the serum and body fluid (such as saliva, sputum and the like) related to tumor organs.

Currently, methylation of ctDNA based on bisulfite conversion is the most widespread research method for ctDNA, but ctDNA is easily fragmented after bisulfite treatment and cannot be completely converted. In addition, previous techniques have often used single or multiple PCR to detect single or small numbers of methylated sites, which is limited in throughput. Therefore, it is of great importance to develop a method for detecting methylation sites in thousands of regions at the same time in a high throughput and with high sensitivity.

Disclosure of Invention

The invention aims to provide a method for detecting tumor ctDNA methylation with high flux and high sensitivity, which is applied to the next generation sequencing process, has the advantages of high flux, digital analysis and high sensitivity, and can be well applied to the determination of the ctDNA methylation in cfDNA.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for detecting tumor ctDNA methylation in high throughput and high sensitivity comprises the following steps:

s1, screening an area with methylation state difference between a tumor and a normal tissue, and designing and preparing a biotin-modified DNA single-strand hybridization capture probe library aiming at CpG point positions of the screened area;

s2, adding common joints at two ends of the DNA of the blood sample to be detected, and adopting methylation insensitive endonuclease to recognize and enzyme-cut the unmethylated DNA double-chain molecule containing the enzyme cutting sites;

s3, carrying out PCR amplification on DNA by using a common primer, and hybridizing and enriching a target region by using the biotin-modified DNA single-stranded hybridization capture probe library prepared in S1;

s4, carrying out PCR amplification on the enriched product, preparing a sequencing library, and carrying out high-throughput sequencing to determine a DNA fragment sequence;

s5, comparing the sequencing depth of the site contrast without enzyme digestion or enzyme digestion with the sequencing depth of the target region after enzyme digestion to calculate the proportion of DNA molecules of the target region which are not subjected to enzyme digestion, thereby calculating the proportion of DNA methylation of the region.

In order to distinguish methylation state, the invention adopts methylation insensitive endonuclease capable of recognizing CpG sites, which can recognize and cut specific DNA sequence, usually 4-8 bases. Typically the cleavage site is in the recognition site, and there are recognition sites in the vicinity of the cleavage site. When the C at a CpG site is methylated, these methylation insensitive endonucleases are not able to cut recognition sites or cleavage sites. The methylated DNA molecules thus remain intact, while the unmethylated DNA is cut into 2 pieces. For example, the restriction enzyme HpaII recognizes CCGG, Hin6I recognizes GCGC, SmaI recognizes CCCGGG and generates CCCGGG cleavage sites; when the site C in CG is methylated in these recognition sequences, it cannot be cleaved, whereas when C in CG is not methylated, it can be cleaved. The digested DNA fragment can not be used as a template molecule to be amplified by PCR; whereas DNA that has not been cleaved by enzyme can be amplified by PCR. And then, comparing the sequencing depth of the sites which are not digested or are not digested with the sequencing depth of the target region after digestion to calculate the proportion of the DNA molecules of the target region which are not digested, thereby calculating the proportion of the DNA methylation of the region.

Since human genomic DNA is very large, it is costly if the entire genome is analyzed. This requires analysis of regions of the human genome where there is a difference in DNA methylation. To remove non-target regions, the selected regions were mostly significantly different in DNA methylation in tumor tissue and corresponding normal tissue. The invention screens restriction enzyme cutting site area which is not sensitive to methylation and a small part of area without cutting site (as internal reference) as target area, when preparing probe, it can adopt method of using terminal transferase and biotin modified nucleotide substrate after DNA synthesis single strand, such as biotin modified dNTP, NTP, ddNTP, NTP is ATP, CTP, GTP, TTP or UTP; the method of chip synthesis can also be used, and the length of the synthesized probe is generally 20 bases to 170 bases, and the optimal length is 70-120 bases. The invention synthesizes and prepares a probe library containing 624 biotin-modified DNA single-stranded hybrid capture probes aiming at a selected target region (see Table 2 in detail). Wherein, one part of the probe coverage area is provided with CCGG enzyme cutting sites, and one part of the probe coverage area is not provided with CCGG enzyme cutting sites as an internal reference and is not cut by selected restriction enzymes; therefore, the number of DNA fragments (peak height) of the genome does not change after the cleavage, while the DNA fragments in the region having the cleavage site change (decrease) after the cleavage, and the methylation at the CG site inhibits the cleavage. Thus, these regions with cleavage sites can be compared to the peak height relative to the internal reference to calculate the proportion of methylation.

The invention uses probe pool to do hybridization to enrich the DNA of the target area. The process comprises the steps of performing end repair, A addition, public junction connection and the like on genomic DNA (including ctDNA); and then carrying out enzyme digestion by methylation insensitive restriction enzyme, and carrying out PCR amplification on the product after enzyme digestion by using a common primer. The amplified PCR product was hybridized with biotin-modified probe. The hybridization conditions are generally 4-60 hours at 45-70 degrees, and the optimal hybridization is stable at 55-65 degrees. When hybridizing, adding a joint blocking sequence C0t-1DNA for blocking. The genomic DNA library for probe hybridization is hybridized by using 100ng-10000ng, generally 500ng-3000ng of DNA library; the amount of probe is 0.1ng-100ng, and the optimal amount is 2-20 ng. And capturing a hybrid compound generated by hybridization by using streptavidin magnetic beads, performing denaturation elution to obtain an enriched target DNA fragment, and performing PCR amplification by using a common primer to obtain a sequencing library. And identifying the quantity of the DNA fragments in the target region through high-throughput sequencing. Thus, the DNA fragments in the target area are enriched, the cost is saved, and the analysis sensitivity is improved.

In the present invention, the genomic DNA to be tested may be human genomic DNA, and may be blood-free DNA (cfDNA), paraffin-embedded tissue DNA (FFPE DNA), or blood-free circulating tumor tissue DNA (ctDNA). The size of the DNA fragment may be 0.01-0.25kb, 0.01-0.3kb, 0.01-0.4kb, 0.01-0.5kb, 0.02-0.25kb, 0.01-5kb, 0.1-1kb, 0.1-5kb, 0.2-0.4kb, 0.5-1kb, 1-2kb, 2-3kb, 3-4kb, 4-5kb, and when the genomic DNA to be tested is circulating tumor DNA (ctDNA), the size of the fragment is generally about 170 bp.

Of course, the probe library disclosed by the invention, the detection kit prepared from the probe library provided by the invention, and the application of the probe library disclosed by the invention in the preparation of the detection kit for detecting the tumor ctDNA methylation with high flux and high sensitivity also belong to the protection contents of the invention.

The invention has the beneficial effects that:

1. the method of the invention introduces 2 public sequencing joint sequences to the DNA of a blood sample to be detected, utilizes methylation insensitive endonuclease to carry out enzyme digestion, so that methylated DNA fragments are reserved, target fragments are enriched by probe hybridization, and then the methylation state of the target fragments is calculated by high-throughput sequencing analysis. First, the present invention uses methylated restriction enzymes to remove unmethylated DNA while retaining methylated DNA, allowing the methylated DNA molecules to be enriched, thereby increasing sensitivity. Secondly, the invention adopts an endonuclease method for enzyme digestion, the reaction condition is mild, large-scale DNA degradation cannot be caused, and the sensitivity is high. Thirdly, the invention enriches the target region DNA by adopting the probe target capture, thereby reducing the cost in a limited way. In addition, the method is equivalent to a methylation analysis technology based on PCR, hundreds of sites can be analyzed simultaneously, the flux is high, and the sensitivity is improved when a large number of sites are analyzed simultaneously. In addition, the invention adopts high-throughput sequencing technology to analyze the enriched fragments, and can digitally evaluate the DNA methylation state.

2. The method provided by the invention can be well applied to the determination of the methylation of the ctDNA in the cfDNA. Because the amount of cfDNA obtained from blood is small, the bisulfite treatment is easy to cause a great amount of DNA degradation, and the ctDNA is easy to fragment after the bisulfite treatment and can not be completely converted, the method of the invention avoids the problems of DNA great degradation, ctDNA fragmentation and incomplete conversion caused by the sulfite treatment, and therefore, the method of the invention can be well applied to the determination of the methylation of the ctDNA in the cfDNA.

Drawings

FIG. 1 is a schematic flow chart of the present invention;

FIG. 2 is a result chart of the digestion time and digestion efficiency of HpaII;

FIG. 3 is a diagram showing the results of sequencing before and after digestion and the results of sulfite-specific primer verification.

Detailed Description

The present invention will now be described more fully hereinafter with reference to the accompanying specific embodiments, in which some, but not all embodiments of the invention are shown. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments disclosed herein are intended to be within the scope of the present invention.

Example 1

The invention discloses a method for detecting tumor ctDNA methylation with high flux and high sensitivity, a technical flow schematic diagram is shown in figure 1, and the specific embodiment is as follows:

firstly, preparing a DNA sample library

1. Blood free dna (cfdna) extraction: collecting fresh blood with EDTA-Na + tube by using free DNA extraction kit of magenta, 7-8 ml; centrifuging at room temperature (1000g/min,10min), collecting supernatant, centrifuging at 4 deg.C, 12000 r/min, 10min, and extracting cfDNA from the obtained supernatant (4-5 ml) according to kit instructions.

cfDNA end repair: the end repair of the DNA fragment can be performed using Klenow fragment having 5 '-3' polymerase activity and 3 '-5' polymerase activity but lacking 5 '-3' exonuclease activity, T4 DNA polymerase and T4 polynucleotide kinase. Therefore, the DNA fragment can be conveniently and accurately subjected to end repair. For the convenience of subsequent processing, a step of purifying the DNA fragment subjected to end repair can be further included. The DNA was filled in at the 5 'protruding sticky ends and blunt-ended at the 3' protruding sticky ends using T4 polymerase and Klenow E.coli polymerase fragments to generate blunt ends for subsequent blunt end ligation. The reaction was carried out in a PCR amplification apparatus at 20 ℃ for 30 minutes. The DNA was purified by column chromatography using a purification kit from commercial company.

Reactive materials	Volume of
		Purified DNA sample library	50 microliter
Phosphorylation reaction buffer	10 microliter
		Deoxybase mixture dNTPs (10mM each)	4 microliter
T4 DNA polymerase	5 microliter
		Klenow E.coli polymerase fragment	1 microliter
T4 polynucleotide kinase	5 microliter
		Nuclease-free water	The total volume is supplemented to 100 microliter

3. Adding base A to the 3' end of the DNA sample

A base A is added to the 3' -end of the DNA fragment subjected to end repair so as to obtain a DNA fragment having a cohesive end A. In order to accurately add the base A to the 3 '-end of the end-repaired DNA fragment, the base A can be added to the 3' -end of the end-repaired DNA fragment by using Klenow (3 '-5' exo-), that is, Klenow having 3 '-5' exonuclease activity. For convenience of subsequent processing, a step of purifying the DNA fragment having the sticky end A may be further included. The reaction was carried out in a PCR amplification apparatus at 37 ℃ for 30 minutes. The DNA was purified by column chromatography using a purification kit from commercial company.

Reactive materials	Volume of
		Purified DNA sample library	About 30 microliters
10 XKlenow E.coli polymerase buffer	5 microliter
		Deoxybase dATP (10mM)	2.5 microliter
Klenow E.coli polymerase fragment	3 microliter
		Nuclease-free water	The total volume is compensated to 50 microliter

4. Adding linkers at two ends of the DNA, wherein the linker sequence is shown in the sequence table 1, the linkers are a pair of DNAs (one of which has phosphorylation at 5' end), and the double strands of the DNAs are formed after annealing (such as AdA-1 and ADBL-1 annealing); the DNA was purified on a column using a commercial purification kit, reacted at 16 ℃ for 8h, and then purified using 1.8-fold XP beads.

Reactive materials	Volume of
		Purified DNA sample library	About 15 microliters
2 XT 4 DNA ligase buffer	5 microliter
		DNA two-end connector (25uM)	6 microliter
T4 DNA ligase	3 microliter
		Nuclease-free water	The total volume is compensated to 50 microliter

5. The enzyme was digested with Hpa II as follows:

reactive materials	Volume of
		Purified adaptor-ligated DNA	About 21 microliters
10×cutsmart buffer	2.5 microliter
		Hpa II enzyme (NEB product)	2 microliter

After 3h reaction at 37 ℃ the mixture was purified by 1.8 XXP beads, the digestion time and efficiency are shown in FIG. 2.

6. Amplification of DNA templates

Taking the sample as a plasma sample containing a trace amount of free DNA fragments as an example, the first amplification allows the amount of nucleic acids to meet the requirement of chip/probe hybridization capture because it contains an extremely trace amount of target free DNA fragments. The primers used contained an index (see Table 1) so that multiple samples could be mixed for hybridization.

Index primers, Index sequences and adapter sequence Listing (Table 1)

7. Polymerase Chain Reaction (PCR), performed in a PCR amplificator.

PCR conditions were as follows: placing in a PCR amplification instrument, pre-denaturing at 98 deg.C for 30 s, annealing at 65 deg.C for 30 s, extending at 72 deg.C for 30 s, circulating 6-8 times (DNA sample bank), and extending at 72 deg.C for 5 min.

The PCR amplification product was purified using the Beckman Coulter Ampure XP Beads kit (cat # A63881).

8. Quality detection of amplified sample library

And (3) carrying out qualitative and quantitative analysis on the DNA by using a biological analyzer, and confirming that the length peak value of the purified fragment is reasonable, about 300-400 bp. Or 2% agarose gel electrophoresis is used for DNA fragment size analysis, and the size of the DNA sample library fragment is reasonable, about 300-400 bp.

DNA qualitative and quantitative analysis was performed using a Bio-Fragment Analyzer bioanalyzer, and the peak of the Fragment length after purification was confirmed to be reasonable, about 300 and 400 bp.

Secondly, hybridizing the DNA sample library with the probe library

Note: and the index is the library index of the sample, and the corresponding index is added.

And (3) uniformly mixing the hybridization reaction solution, placing the mixture into a PCR amplification instrument for reaction under the conditions of 95 ℃ and 2 minutes, immediately placing the mixture on ice and keeping the mixture for 2 minutes after the reaction is finished, and then placing the mixture into a 56 ℃ constant-temperature hybridization instrument for hybridization, wherein the hybridization time is 6-48 hours.

Probe library sequence table (Table 2)

Thirdly, obtaining the methylated DNA fragments enriched by hybridization

1. Preparation of streptavidin magnetic beads

Dynabeads M-270 streptavidin (Invitrogen) streptavidin magnetic beads or other commercial company streptavidin magnetic beads were used. And (5) placing the magnetic beads on a blending machine for blending.

And (3) washing magnetic beads: 50 microliters of the Beads were washed once with a Beads Wash Buffer (1M NaCl, 10mM Tris-HCl pH8.0, 10mM EDTA pH8.0, 0.1% Triton X-100), the supernatant was removed by magnet adsorption, and then suspended in 50 microliters of the Beads Wash Buffer for use.

2. Capturing, washing and eluting DNA

Capturing: the suspension of magnetic beads obtained above was added to the hybridization mixture that had been hybridized for 8 hours, and the DNA was captured by rotation in a constant temperature hybridization apparatus at 56 ℃ for 45 minutes, and the supernatant was removed by the action of a magnetic field.

Washing: adding 150 microliters of Beads Wash Buffer to resuspend the magnetic Beads, adsorbing the supernatant by a magnet, washing the magnetic Beads with the captured DNA by using 100 microliters of Beads Wash Buffer at 37 ℃ for 15 minutes, and adsorbing the supernatant by the magnet; adding a small amount of Wash buffer I (1 XSSC, 0.1% SDS) to suspend the magnetic beads, changing the tube to a 1.5 ml centrifuge tube, adding the Wash buffer I to 500 microliters, and spin-washing the magnetic beads with captured DNA at 37 ℃ for 15 minutes; then adsorbing the supernatant by a magnet, and repeatedly washing the magnetic beads once by using Wash buffer I; adding a small amount of Wash buffer II (0.1 XSSC, 0.1% SDS) to suspend the magnetic beads, changing the tube to a new 1.5 ml centrifuge tube, adding the Wash buffer II to 500. mu.l, rotating and washing the magnetic beads with the captured DNA at 56 ℃ for 15 minutes (note: washing the magnetic beads by the Wash buffer II is repeated for 1 time, and the time is 15 minutes respectively), and then adsorbing the supernatant by a magnet; the beads were washed briefly with 50. mu.l Washing buffer III (10mM Tris-HCl pH8.0, 0.1% Triton X-100) mix, transferred to a 200. mu.l PCR tube, and the supernatant was removed by magnetic adsorption.

And (3) elution: resuspend the captured DNA beads in 50. mu.l nuclease-free water, react the suspension in a PCR instrument at 95 ℃ for 4 minutes, immediately adsorb the beads at high temperature, and take the supernatant, which contains the enriched methylated DNA fragments.

Fourth, PCR amplification, purification and quality detection

1. The enriched methylated DNA library is further amplified in preparation for loading on a sequencing instrument.

Reactive materials	Volume of
		Purified adaptor-ligated DNA	About 21 microliters
2X high fidelity DNA polymerase mix	25 microliter
		P5_primer(25uM)	2 microliter
P7_primer 25uM)	2 microliter
			The total volume is compensated to 50 microliter

PCR conditions were as follows: pre-denaturation at 98 ℃ for 2 minutes, denaturation at 98 ℃ for 15 seconds, annealing at 60 ℃ for 20 seconds, extension at 72 ℃ for 20 seconds, circulating for 20-22 times, and finally extension at 72 ℃ for 5 minutes.

2. PCR amplification products were purified using the Beckman Coulter Ampure Beads kit (cat # A63881).

3. And (5) quality detection of the enriched DNA sample library after amplification.

And (3) carrying out DNA fragment size analysis by using 2% agarose gel electrophoresis, wherein the result is shown in figure 2, and the amplified enriched DNA sample library fragment has reasonable size of 300-400 bp.

4. And (5) sequencing. The library was high throughput sequenced using the Illumina sequencing platform Nova-seq or Hiseq platform, with PE150 mode (end-pairing mode) per sample and sequencing throughput of approximately 2G/sample.

And (3) displaying the result:

comparative example 1:

the other conditions were not changed, except that no digestion was performed. The results show that the PCR product was completely cleaved 3 hours after the digestion with HpaII; the PCR without enzyme cleavage showed a high capillary electrophoresis peak height as shown in FIG. 2.

Comparative example 2

Sequencing was performed using a conventional sulfite-specific primer method. The results of sequencing by restriction, sequencing by restriction and sequencing by sulfite-specific primer were compared, and the results are shown in FIG. 3. The result shows that the region is completely cut off after HpaII enzyme digestion, which indicates that the region is not methylated; the sequencing result by using a sulfite specific primer method also shows that the region is not methylated, and the result is completely consistent with the result of the method.

Claims

1. A method for detecting tumor ctDNA methylation in high throughput and high sensitivity is characterized by comprising the following steps:

s1, screening an area with methylation state difference between a tumor and a normal tissue, and designing and preparing a capture probe library aiming at CpG point positions of the screened area;

s3, carrying out PCR amplification on DNA by using a common primer, and capturing an enriched target region by using a capture probe library prepared in S1;

2. The method for high-throughput and high-sensitivity detection of tumor ctDNA methylation according to claim 1, wherein the screening region comprises a region containing a methylation-insensitive endonuclease cleavage site and a region serving as an internal reference and not containing a methylation-sensitive endonuclease cleavage site.

3. The method for high-throughput and high-sensitivity detection of tumor ctDNA methylation according to claim 2, wherein the methylation-insensitive endonuclease is an enzyme that can recognize a specific sequence containing a CG site and cleave the site, but cannot cleave the site when the site is methylated.

4. The method for high-throughput and high-sensitivity detection of tumor ctDNA methylation according to claim 3, wherein the methylation-insensitive endonuclease is a restriction endonuclease HpaII, Hin6I, or SmaI.

5. The method for high-throughput and high-sensitivity detection of tumor ctDNA methylation according to claim 4, wherein the capture probe library designed and prepared in S1 is a biotin-modified DNA single-stranded hybrid capture probe library, the probe length of the capture probe library is 20-170 bases, and the biotin modification is performed at the 3-terminal or 5-terminal middle position of the DNA.

6. The method for high-throughput and high-sensitivity detection of tumor ctDNA methylation according to claim 5, wherein the capture probe library designed and prepared in S1 is a biotin-modified DNA single-stranded hybrid capture probe library with a probe length of 70-120 bases.

7. The method for high-throughput and high-sensitivity detection of tumor ctDNA methylation according to claim 6, wherein the capture probe library designed and prepared in S1 comprises 624 probe sequences, and the nucleotide sequences of the 624 probe sequences are shown in SEQ ID Nos. 1-624.

8. A probe library for detecting tumor ctDNA methylation with high flux and high sensitivity is characterized by comprising probes covering a methylation insensitive endonuclease enzyme cutting site region and probes not covering a methylation sensitive endonuclease enzyme cutting site.

9. The probe bank for detecting the tumor ctDNA methylation with high throughput and high sensitivity according to claim 8, wherein the probe bank comprises 624 probe sequences, and the nucleotide sequences of the 624 probe sequences are shown in SEQ ID Nos. 1 to 624.

10. The detection kit prepared from the probe library for detecting the tumor ctDNA methylation in high throughput and high sensitivity according to any one of claims 8 to 9.