WO2019024404A1 - Genetic marker for detecting pancreatic cancer, kit, and pancreatic cancer detection method - Google Patents

Abstract

The present invention provides a genetic marker for detecting pancreatic cancer, a kit, and a pancreatic cancer detection method The genetic marker comprises one or more of the following genes: maltase, C-type exogenous lectin family 4 member C, sorting protein 7, interstitial homeobox 2, FAT atypical cadherin 1, flavin-contained monooxygenase 3, cystic fibrosis transmembrane conductance regulator protein, phospholipid-phosphatase-associated protein 3, α albumin and collagen V-type α2 chain; the content of 5-hydroxymethylcytosine in the genetic marker of the pancreatic cancer is detected by means of a high-throughput sequencing, so as to determine whether the pancreatic cancer exists.

Description

Gene markers, kits and pancreatic cancer detection methods for detecting pancreatic cancer Technical field

The present invention relates to the field of clinical molecular diagnosis of pancreatic cancer. In particular, the present invention relates to a genetic marker, a kit, and a pancreatic cancer detecting method for detecting pancreatic cancer.

Background technique

Pancreatic cancer (PCA) is a malignant tumor of the digestive tract that is highly malignant and difficult to diagnose and treat. About 90% of ductal adenocarcinomas originate from the ductal epithelium. The incidence of this disease is higher in men than in women. The ratio of male to female is 1.5 to 2:1. Male patients are far more common than premenopausal women. The incidence of postmenopausal women is similar to that of males. The cause of pancreatic cancer is not yet fully understood. Its occurrence is related to smoking, drinking, high-fat and high-protein diet, excessive coffee consumption, environmental pollution and genetic factors; recent investigations have found that the incidence of pancreatic cancer in diabetic population is significantly higher than that of the general population; Patients with pancreatitis have a certain relationship with the incidence of pancreatic cancer, and the proportion of pancreatic cancer in patients with chronic pancreatitis has increased significantly. In recent years, the incidence of pancreatic cancer has increased significantly. Since the 1930s, the incidence of PCA in the United States, Britain, Japan and other countries has increased by 2 to 4 times. The incidence of PCA in malignant tumors in Shanghai increased from 14th in 1974 to 8th in 1993. The rate of increase in PCA is second only to pancreatic cancer, with an increase of about 1.5% every 10 years.

Although, in recent years, the development of abdominal surgery has been changing with each passing day. The level of diagnosis and treatment of many abdominal diseases, including tumors, has been greatly improved with the advancement of basic medicine, the improvement of imaging techniques and the application of molecular diagnostic techniques. However, the progress in the diagnosis and treatment of pancreatic cancer is not satisfactory. PCA is insidious, with no specific symptoms, often accompanied by early spread and metastasis, and is one of the worst prognosis. Eighty-five percent of patients with pancreatic cancer are in advanced stage at the time of treatment, and only about 20% of them are eligible for surgery. The annual PCA death to morbidity ratio is 0.99. The five-year survival rate of the confirmed patients is 1.3%, and the average median survival time is only 4.1 months. It is called the “stubborn fortress of the 21st century medicine”.

At present, the detection of pancreatic cancer mainly through imaging, tissue biopsy, serological testing and the like. However, imaging is susceptible to operator experience and relies on equipment, which is expensive, especially in the case of limited medical resources, its accuracy is difficult to guarantee, it is difficult to widely and routinely apply, and CT and ultrasound are difficult to diagnose less than 2cm. Pancreatic tumors. Tissue biopsy is the gold standard for clinically diagnosed pancreatic cancer, but there are many limitations in tissue biopsy, such as the difficulty of surgical sampling, or the inconvenience of puncture in some cancer sites, and the puncture itself will bring certain clinical risks. Puncture screening can cause great pain to patients. The most widely used serological test is the detection of carcinoembryonic antigen (CEA), but the sensitivity and specificity of CEA for early pancreatic cancer are not high, and often increase after tumor metastasis.

Therefore, the search for new markers of pancreatic cancer, especially for early warning and early diagnosis, is of great significance in improving the diagnosis rate of early pancreatic cancer, achieving early intervention and reducing the mortality rate of pancreatic cancer.

Summary of the invention

The present invention unexpectedly finds a plurality of highly informative information by performing high-throughput sequencing on normal samples and pancreatic cancer samples, and analyzing the content of 5-hydroxymethylcytosine (5-hmC) in each gene. A genetic marker that can be used to detect pancreatic cancer.

Accordingly, a first aspect of the invention relates to a genetic marker for detecting pancreatic cancer comprising one or more of the following genes: maltase (SI), C-type lectin family 4 member C (CLEC4C), sub- Protein 7 (SNX7), mesenchymal homeobox 2 (MEOX2), FAT atypical cadherin 1 (FAT1), flavin containing monooxygenase 3 (FMO3), cystic fibrosis transmembrane conductance regulatory protein (CFTR) ), phospholipid phosphatase-related protein 3 (PLPPR3), alpha albumin (AFM), and collagen V-type α2 chain (COL5A2). Preferably, the genetic markers include SI, CLEC4C, SNX7, MEOX2, FAT1, FMO3, CFTR, PLPPR3, AFM and COL5A2.

The present invention also relates to the use of the above-mentioned gene marker for detecting pancreatic cancer, and detecting the content of 5-hydroxymethylcytosine in the pancreatic cancer gene marker by high-throughput sequencing, thereby determining whether pancreatic cancer is present.

A second aspect of the invention relates to a method for detecting pancreatic cancer comprising the steps of:

a) determining the 5-hmC content of the genetic markers of the invention in normal and subject samples;

b) normalizing the 5-hmC content of the corresponding gene marker in the sample of the subject using the 5-hmC content of the gene marker in the normal sample as a reference;

c) mathematically correlating the 5-hmC content of the standardized genetic marker and obtaining a score;

d) Obtain a test result based on the score.

In one embodiment, the sample is a free DNA fragment in a subject or normal human body fluid, or is derived from intact genomic DNA in organelles, cells, and tissues. Among them, body fluids are blood, urine, sweat, sputum, feces, cerebrospinal fluid, ascites, pleural effusion, bile, pancreatic juice, and the like.

In one embodiment, the 5-hmC content of the genetic markers of the invention can be determined by any method known to those skilled in the art, including, for example, but not limited to, glucosylation, restriction endonucleases Method, chemical labeling method, precipitation method combined with high-throughput sequencing method, single molecule real-time sequencing method (SMRT), oxidized bisulfite sequencing method (OxBS-Seq), and the like. The principle of the glucosylation method is to transfer glucose to the hydroxyl group in the presence of glucose donor substrate uridine nucleoside diphosphate glucose (UDP-Glu) using T4 phage β-glucose transferase (β-GT). β-Glucosyl-5-hydroxymethylcytosine (5-ghmC) was produced. Isotopically labeled substrates can also be used for quantification. The restriction endonuclease method and the chemical labeling method were further developed on the basis of the glucosylation method. The principle of the restriction endonuclease method is that the glucosylation reaction changes the enzymatic cleavage properties of some restriction enzymes. The methylation-dependent restriction enzymes MspI and HpaII recognize the same sequence (CCGG), but their sensitivity to methylation status is different: MspI recognizes and cleaves 5-methylcytosine (5-mC) And 5-hmC, but not 5-ghmC; HpaII only cleaves completely unmodified sites, and any modification on cytosine (5-mC, 5-hmC, 5-ghmC) blocks cleavage. If the CpG site contains 5-hmC, the band can be detected after glycosylation and enzymatic hydrolysis, and there is no band in the unglycosylated control reaction; qPCR can also be used for quantitative analysis. In addition, other restriction enzymes also have a hindrance to 5-ghmC digestion, and can be applied to 5-hmC detection (eg, GmrSD, MspJI, PvuRts1I, TaqI, etc.). The principle of the chemical labeling method is to chemically modify the glucose on the substrate of the enzyme reaction into UDP-6-N3-glucose, and transfer 6-N3-glucose to the position of the hydroxymethyl group to form N3-5ghmC. Subsequently, a single molecule of biotin was added to each 5-hmC by click chemistry, combined with next-generation high-throughput DNA sequencing technology or single-molecule sequencing technology to analyze the distribution of 5-hmC in genomic DNA. The precipitation method is to modify 5-hmC in a special way and then specifically capture it from genomic DNA and perform sequencing analysis. Oxidized bisulfite sequencing is the first method to quantify 5-hmC with single base resolution. Firstly, 5-hmC is subjected to KRuO4 oxidation treatment to produce 5-formylcytosine (5fC), and then heavy Sulfite sequencing. In this process, 5-hmC is first oxidized to 5fC, and then deaminated to form U. Usually, quantitative detection of 5-hmC is performed simultaneously using a variety of detection methods.

In one embodiment of the invention, the 5-hmC content of the genetic markers of the invention is determined using chemical labeling in conjunction with high throughput sequencing. In this particular embodiment, the method of determining the 5-hmC content of a genetic marker of the present invention comprises the steps of: fragmenting DNA from a sample of a pancreatic cancer patient and a normal human; repairing the fragmented DNA end And finishing the end; the end-filled DNA is ligated to the sequencing linker to obtain a ligation product; the 5-hydroxymethylcytosine in the ligation product is labeled by a labeling reaction; enrichment contains a 5-hydroxymethylcytosine tag The DNA fragment is obtained as an enriched product; the enriched product is subjected to PCR amplification to obtain a sequencing library; the sequencing library is subjected to high-throughput sequencing to obtain a sequencing result; and the genetic content of 5-hydroxymethylcytosine is determined according to the sequencing result. . Wherein the labeling reaction comprises: i) covalent attachment of a sugar having a modifying group to a methylol group of 5-hydroxymethylcytosine using a glycosyltransferase, and ii) direct or indirect attachment of biotin Click on the chemical substrate to react with 5-hydroxymethylcytosine with a modifying group. Wherein step i) and step ii) may be carried out sequentially or simultaneously in one reaction. This labeling method reduces the amount of sample required for sequencing, and the biotin tag on 5-hydroxymethylcytosine allows it to display higher kinetic signals in sequencing, improving the accuracy of nucleotide recognition. In this embodiment, the glycosyltransferase includes, but is not limited to, T4 phage β-glucosyltransferase (β-GT), T4 bacteriophage α-glucosyltransferase (α-GT), and the same Or a similarly active derivative, analog, or recombinase; the saccharide with a modifying group includes, but is not limited to, a saccharide with an azide modification (eg, 6-N3-glucose) or with other chemical modifications a saccharide (e.g., a carbonyl group, a thiol group, a hydroxyl group, a carboxyl group, a carbon-carbon double bond, a carbon-carbon triple bond, a disulfide bond, an amine group, an amide group, a diene, etc.), among which a saccharide modified with azide is preferred. The chemical group for indirectly linking the biotin and the click chemical substrate includes, but is not limited to, a carbonyl group, a thiol group, a hydroxyl group, a carboxyl group, a carbon-carbon double bond, a carbon-carbon triple bond, a disulfide bond, an amine group, Amido group, diene. In this embodiment, the DNA fragment containing the 5-hmC label is preferably enriched by a solid phase material. Specifically, a DNA fragment containing a 5-hydroxymethylcytosine label can be bound to a solid phase material by a solid phase affinity reaction or other specific binding reaction, and then the unbound DNA fragment can be removed by multiple washings. Solid phase materials include, but are not limited to, silicon wafers or other chips with surface modification, such as artificial polymer beads (preferably 1 nm to 100 um in diameter), magnetic beads (preferably 1 nm to 100 um in diameter), agarose beads, etc. (Preferably from 1 nm to 100 um in diameter). The washing liquid used for solid phase enrichment is a buffer well known to those skilled in the art, including but not limited to: containing Tris-HCl, MOPS, HEPES (pH=6.0-10.0, concentration between 1 mM and 1 M), NaCl (0) - 2M) or a buffer such as Tween 20 (0.01% - 5%). In this embodiment, PCR amplification is preferably performed directly on the solid phase to prepare a sequencing library. If necessary, after performing PCR amplification on a solid phase, the amplified product can be recovered and subjected to a second round of PCR amplification to prepare a sequencing library. The second round of PCR amplification can be performed using conventional methods known to those skilled in the art. Optionally, one or more purification steps may be further included in the process of preparing the sequencing library. Any purification kit known or commercially available to those skilled in the art can be used in the present invention. Purification methods include, but are not limited to, gel electrophoresis gel recovery, silica gel membrane spin column method, magnetic bead method, ethanol or isopropanol precipitation method, or a combination thereof. Optionally, the sequencing library is quality checked prior to high throughput sequencing. For example, the library is subjected to fragment size analysis and the concentration of the library is absolutely quantified using the qPCR method. Sequencing libraries that pass quality checks can be used for high throughput sequencing. Then, a certain number (1-96) of libraries containing different barcodes were mixed at the same concentration and sequenced according to the standard on-line method of the second generation sequencer to obtain sequencing results. Various second generation sequencing platforms and related reagents known in the art can be used in the present invention.

In one embodiment of the invention, the sequencing results are preferably aligned with a standard human genome reference sequence, and the sequences in which the gene markers of the invention are aligned are selected, ie, the alignment sites and gene features (eg, groups) are selected. The number of reads of the coincident region of the protein modification site, transcription factor binding site, gene exon intron region, and gene promoter, etc., to represent the level of modification of 5-hmC on the gene, thereby determining 5-hmC The amount on the genetic marker. Preferably, the sequencing results are first cleared of low-quality sequencing sites prior to the alignment, wherein factors that measure the quality of the sequencing sites include, but are not limited to, base quality, reads mass, GC content, repeat sequences, and number of Overrepresented sequences. The various alignment software and analytical methods involved in this step are known in the art.

In one embodiment of the present invention, determining the 5-hmC content of the gene marker means determining the 5-hmC content of the full length of the gene marker or determining the 5-hmC content of a fragment of the gene marker or combination.

According to the present invention, after determining the 5-hmC content of each gene marker, the 5-hmC content of the corresponding gene marker in the sample of the subject is used as a reference with the 5-hmC content of the gene marker in the normal sample. standardization. For example, the 5-hmC content of the same gene marker in the normal sample and the subject sample is X and Y, respectively, and the normalized 5-hmC content of the genetic marker in the subject sample is Y/X.

According to the present invention, after the data is normalized, the standardized 5-hmC content of each gene marker is mathematically correlated to obtain a score, thereby obtaining a detection result based on the score. As used herein, "mathematical association" refers to any computational or machine learning method that correlates the 5-hmC content of a genetic marker from a biological sample with the diagnosis of pancreatic cancer. One of ordinary skill in the art understands that different computing methods or tools can be selected to provide the mathematical associations of the present invention, such as elastic network regularization, decision trees, generalized linear models, logistic regression, highest score pairs, neural networks, linear and Quadratic Discriminant Analysis (LQA and QDA), Naive Bayes, Random Forest, and Support Vector Machines.

In one embodiment of the present invention, the specific steps for mathematically correlating the standardized 5-hmC content of each gene marker and obtaining a score are as follows: multiplying the normalized 5-hmC content of each gene marker by a weighting coefficient to obtain the gene The predictor of the marker t; the predictor t of each gene marker is added to obtain a total predictor T; the total predictor T is subjected to Logistic conversion to obtain a score P; if P>0.5, the subject sample suffers Pancreatic cancer; if P ≤ 0.5, the subject sample is normal. The weighting factor described herein refers to the art by the art in consideration of factors that may affect the 5-hmC content (eg, subject area, age, sex, below, smoking history, drinking history, family history, etc.) The coefficients obtained by various advanced statistical analysis methods known to the person.

A third aspect of the present invention also relates to a kit for detecting pancreatic cancer using the above gene marker, which comprises a reagent and a specification for measuring a 5-hmC content of the above gene marker. Agents for determining the 5-hmC content of a genetic marker are known to those skilled in the art, such as T4 bacteriophage beta-glucose transferase and isotopic labeling (for glucosylation), restriction enzymes (for restriction) Endonuclease method), glycosyltransferase and biotin (for chemical labeling), reagents for PCR and sequencing, and the like.

Compared to the prior art, the method for detecting pancreatic cancer of the present invention is based on the 5-hmC content of the gene marker, and thus a wider range of DNA sample sources can be used. Therefore, the method for detecting pancreatic cancer of the present invention has the following advantages: (1) safe and non-invasive, even if the asymptomatic population has high acceptance of the test; (2) the DNA source is extensive, and there is no blind spot in the imaging; 3) High accuracy, high sensitivity and specificity for early pancreatic cancer, suitable for early screening of pancreatic cancer; (4) Convenient operation, good user experience, and easy dynamic monitoring of pancreatic cancer recurrence and metastasis. The gene markers of the present invention can be combined with other clinical indicators to provide more accurate judgments for pancreatic cancer screening, diagnosis, treatment and prognosis.

DRAWINGS

Figure 1 is a graph depicting a pancreatic cancer sample and a healthy sample control of the present invention.

Detailed ways

The present invention will be described in detail below with reference to the embodiments and the accompanying drawings, in which FIG.

Example 1. Screening of pancreatic cancer gene markers

1) Extraction of plasma DNA:

10 ng of plasma DNA was extracted from samples from 20 pancreatic cancer patients and 20 normal subjects, respectively. This step can be carried out using any method and reagent suitable for extracting plasma DNA well known to those skilled in the art.

2) End the plasma DNA, suspend A and connect to the sequencing linker:

Prepare a reaction mixture containing 50 uL of plasma DNA, 7 uL End Repair & A-Tailing Buffer and 3 uL End Repair & A-Tailing Enzyme mix according to the Kapa Hyper Perp Kit instructions (total volume 60 uL), warm bath at 20 ° C for 30 minutes, then warm bath at 65 ° C 30 minute. The following ligation reaction mixture was placed in a 1.5 mL low adsorption EP tube: 5 uL Nuclease free water, 30 uL Ligation Buffer and 10 uL DNA Ligase. 5 uL of the sequencing adaptor was added to the 45 uL ligation reaction mixture, mixed, heated at 20 °C for 20 minutes, and then kept at 4 °C. The reaction product was purified using Ampure XP beads, and eluted with 20 uL of a buffer containing Tris-HCl (10 mM, pH = 8.0) and EDTA (0.1 mM) to obtain a final DNA-ligated sample.

3) Labeling 5-hydroxymethylcytosine:

Prepare a total of 26 uL of labeling reaction mixture: azide-modified uridine diphosphate glucose (ie UDP-N3-Glu, final concentration 50 uM), β-GT (final concentration 1 uM), Mg2+ (final concentration 25 mM) ), HEPES (pH = 8.0, final concentration of 50 mM) and 20 uL of DNA from the above procedure. The mixture was incubated at 37 ° C for 1 hour. The mixture was taken out and purified with Ampure XP beads to obtain purified 20 uL of DNA.

Then, 1 uL of biotin-containing diphenylcyclooctyne (DBCO-Biotin) was added to the above purified 20 uL DNA, and reacted at 37 ° C for 2 hours, followed by purification with Ampure XP beads to obtain a purified labeled product.

4) Solid phase enrichment of DNA fragments containing labeled 5-hydroxymethylcytosine:

First, prepare the magnetic beads as follows: Take 0.5 uL of C1streptadvin beads (life technology) and add 100 uL of buffer (5 mM Tris, pH = 7.5, 1 M NaCl, 0.02% Tween 20), vortex for 30 seconds, then use 100 uL of washing solution. (5 mM Tris, pH = 7.5, 1 M NaCl, 0.02% Tween 20) Wash the beads 3 times, and finally add 25 uL of binding buffer (10 mM Tris, pH = 7.5, 2 M NaCl, 0.04% Tween 20 or other surfactant) and mix Evenly.

Then, the purified labeled product obtained in the above procedure was added to the magnetic bead mixture, and mixed for 15 minutes in a rotary mixer to sufficiently bind.

Finally, the magnetic beads were washed 3 times with 100 uL of washing solution (5 mM Tris, pH = 7.5, 1 M NaCl, 0.02% Tween 20), the supernatant was removed by centrifugation, and 23.75 uL of nuclease-free water was added.

5) PCR amplification:

To the final system of the above procedure, 25 uL of 2X PCR master mix and 1.25 uL of PCR primers (total volume 50 uL) were added and amplified according to the temperature and conditions of the following PCR reaction cycle:

The amplified product was purified using Ampure XP beads to give a final sequencing library.

6) High-throughput sequencing after sequencing the sequencing library:

The obtained sequencing library was subjected to concentration determination by qPCR, and the DNA fragment size content in the library was determined using Agilent 2100. The sequencing libraries passed the QC were mixed at the same concentration and sequenced using an Illumina Hiseq 4000.

7) Determine the 5-hmC content and weighting factor of each gene marker:

The obtained sequencing results were subjected to preliminary quality control evaluation, and after the low-quality sequencing sites were cleared, the reads that met the sequencing quality standards were compared with the human standard genome reference sequence using the Bowtie 2 tool. The feature counts and HtSeq-Count tools were then used to count the number of reads to determine the 5-hmC content of each gene marker. At the same time, using high-throughput sequencing results, the factors that may affect the 5-hmC content were used as covariates, and the weighting coefficients of each gene marker were obtained by logistic regression and elastic network regularization. The results are shown in Table 1.

Table 1: Average normalized 5-hmC content and weighting coefficient of pancreatic cancer gene markers of the present invention

As described above, the average normalized 5-hmC content refers to the ratio of the average 5-hmC content of the gene marker in the pancreatic cancer sample to the average 5-hmC content of the same gene marker in the normal sample. As can be seen from Table 1, the 5-hmC content of the pancreatic cancer gene marker of the present invention is significantly different between the normal sample and the pancreatic cancer sample, and the 5-hmC content of the remaining gene markers except for CLEC4C and PLPPR3. Significantly increased relative to normal people.

Example 2. Effectiveness of pancreatic cancer gene markers

This example demonstrates the effectiveness of the pancreatic cancer gene marker of the present invention for detecting pancreatic cancer.

The 5-hmC content of the 10 pancreatic cancer gene markers of the present invention in the first 82 samples (41 pancreatic cancers and 41 healthy controls) was determined according to the method of Example 1.

Multiplying the normalized 5-hmC content of each gene marker by the corresponding weighting coefficient of the marker in Example 1, obtaining the predictor t of the gene marker, and then adding the predictor t of each gene marker to obtain The total predictor T, and then the total predictor T is subjected to Logistic conversion according to the following formula to obtain the score P:

If P > 0.5, the subject sample has pancreatic cancer; if P < 0.5, the subject sample is normal.

Figure 1 shows the results of distinguishing the batch of samples in accordance with the method of the present invention. As shown in Figure 1, the method of the invention is capable of achieving 95% sensitivity and 87% specificity.

It should be noted that the above description is only for explaining the technical solutions of the present invention, and is not intended to limit the scope of the present invention. Those skilled in the art can easily modify or replace the technical solutions of the present invention without departing from the present disclosure. The essence and scope of the technical solution of the invention.

Claims (10)

1. The use of gene markers for detecting pancreatic cancer, and detecting the presence of pancreatic cancer by high-throughput sequencing to detect the presence of 5-hydroxymethylcytosine in a pancreatic cancer gene marker, the genetic marker comprising one or two The following genes are: maltase (SI), C-type lectin family 4 member C (CLEC4C), sorting protein 7 (SNX7), mesenchymal homeobox 2 (MEOX2), FAT atypical cadherin 1 ( FAT1), flavin contains monooxygenase 3 (FMO3), cystic fibrosis transmembrane conductance regulator (CFTR), phospholipid phosphatase associated protein 3 (PLPPR3), alpha albumin (AFM), and collagen V-type α2 chain (COL5A2).
2. The use according to claim 1, characterized in that the genetic markers include SI, CLEC4C, SNX7, MEOX2, FAT1, FMO3, CFTR, PLPPR3, AFM and COL5A2.
3. A method for detecting pancreatic cancer, comprising the steps of:

   a) determining the content of 5-hydroxymethylcytosine of the gene markers of claims 1 and 2 in the normal sample and the subject sample;

   b) normalizing the 5-hydroxymethylcytosine content of the corresponding gene marker in the subject sample using the 5-hydroxymethylcytosine content of the gene marker in the normal sample as a reference, normal sample and subject The 5-hydroxymethylcytosine content of the same gene marker in the sample is X and Y, respectively, and the standardized 5-hydroxymethylcytosine content of the gene marker in the sample of the subject is Y/X;

   c) mathematically correlating the 5-hydroxymethylcytosine content of the genetic marker normalized in step b) and obtaining a score P;

   d) Obtaining a test result of whether the subject sample has pancreatic cancer according to the numerical value of the score P.
4. The method according to claim 3, wherein the step a) comprises the steps of: fragmenting DNA from a sample of a pancreatic cancer patient and a normal human; repairing and ending the fragmented DNA end; The complemented DNA is ligated to the sequencing linker to obtain a ligation product; the 5-hydroxymethylcytosine in the ligation product is labeled by a labeling reaction; the DNA fragment containing the 5-hydroxymethylcytosine tag is enriched to obtain an enriched product PCR amplification of the enriched product to obtain a sequencing library; high-throughput sequencing of the sequencing library to obtain sequencing results; determining the genetic content of 5-hydroxymethylcytosine according to the sequencing result.
5. The method according to claim 4, wherein said labeling reaction comprises: i) covalently linking a sugar having a modifying group to a hydroxymethyl group of 5-hydroxymethylcytosine using a glycosyltransferase And ii) reacting a click chemical substrate directly or indirectly with biotin with a 5-hydroxymethylcytosine bearing a modifying group.
6. The method according to claim 3, 4 or 5, wherein the step (a) is for determining the content of 5-hydroxymethylcytosine on the entire length of the gene marker or a fragment thereof.
7. The method according to claim 3, 4 or 5, wherein the step c) comprises the step of multiplying the normalized 5-hydroxymethylcytosine content of each gene marker by a weighting coefficient to obtain a prediction of the genetic marker Factor t; adding the predictor t of each gene marker to obtain a total predictor T; the total predictor T is subjected to Logistic conversion to obtain a score P; if P>0.5, the subject sample has pancreatic cancer; P ≤ 0.5, the subject sample is normal.
8. The method according to claim 3, wherein the sample is a free DNA fragment derived from a normal human or a subject's body fluid, or derived from an intact genomic DNA in an organelle, a cell, and a tissue.
9. The method according to claim 8, wherein the body fluid is blood, urine, sweat, sputum, feces, cerebrospinal fluid, ascites, pleural effusion, bile or pancreatic juice.
10. A kit for detecting pancreatic cancer, comprising:

    a) an agent for determining the 5-hydroxymethylcytosine content of the gene marker of claim 1; and b) instructions;

    The 5-hydroxymethylcytosine content refers to the content of 5-hydroxymethylcytosine on the entire length of the gene marker or a fragment thereof.

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