TW201910774A - Gene marker, reagent group and pancreatic cancer detection method for detecting pancreatic cancer - 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, [alpha] albumin and collagen V-type [alpha]2 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 marker, reagent set for detecting pancreatic cancer, and method for detecting pancreatic cancer

The invention relates to the field of clinical molecular diagnosis of pancreatic cancer. Specifically, the present invention relates to a genetic marker for detecting pancreatic cancer, a reagent set, and a method for detecting pancreatic cancer.

Pancreatic cancer (PCA) is a malignant tumor of the digestive tract with a high degree of malignancy, which is difficult to diagnose and treat. About 90% of them are ductal adenocarcinomas originating from the ductal epithelium. The incidence of this disease is higher in men than in women, with a male-to-female ratio of 1.5 to 2: 1. Male patients are far more common than women before menopause. The incidence of women after menopause is similar to men. The etiology of pancreatic cancer is not yet clear. Its occurrence is related to smoking, alcohol consumption, high-fat and high-protein diet, excessive drinking of coffee, environmental pollution and genetic factors; survey reports in recent years have found that the incidence of pancreatic cancer is significantly higher in the diabetic population than in the general population; others have noted chronic Patients with pancreatitis have a certain relationship with the incidence of pancreatic cancer, and it has been found that the proportion of patients with chronic pancreatitis has a significant increase in pancreatic cancer. In recent years, the incidence of pancreatic cancer has increased significantly. Since the 1930s, the incidence of PCA in the United States, Britain, and Japan has increased by 2 to 4 times. The incidence of PCA in Shanghai from malignant tumors increased from 14th in 1974 to 8th in 1993. The rate of increase in PCA is second only to pancreatic cancer, increasing by about 1.5% every 10 years.

Although, the development of abdominal surgery has changed rapidly in recent years. 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 technology and the application of molecular diagnostic technology. But progress in the diagnosis and treatment of pancreatic cancer has been unsatisfactory. PCA has hidden onset, lacks specific symptoms, and is often accompanied by early spread and metastasis. It is one of the worst prognostic malignancies. 85% of patients with pancreatic cancer are already at the advanced stage of treatment, and only about 20% are feasible for surgical treatment. The death and morbidity ratio of PCA is 0.99 each year, and the five-year survival rate of confirmed patients is 1.3%, with an average median survival time of only 4.1 months. It is known as the "stubborn fortress of 21st century medicine".

At present, the detection of pancreatic cancer is mainly through imaging, histological examination (tissue diagnosis), and serological detection. However, imaging is susceptible to operator experience and is dependent on equipment, which is costly, especially when medical resources are limited, its accuracy is difficult to guarantee, it is difficult to use it widely and routinely, and CT and ultrasound are difficult to diagnose for less than 2 cm. Pancreatic tumor. Histological examination is currently a necessary method for the clinical diagnosis of pancreatic cancer, but histological examination has many limitations, such as difficulty in surgical sampling or inconvenience of puncture in some cancer sites, and the puncture itself will bring certain clinical risks. , Repeated puncture screening will bring great pain to patients. Serum detection is currently the most widely used method for the detection of carcinoembryonic antigen (CEA), but CEA has low sensitivity and specificity for early pancreatic cancer, which often increases after tumor metastasis.

Therefore, finding new pancreatic cancer markers, especially those for early warning monitoring and early diagnosis, is of great significance for improving the diagnosis rate of early pancreatic cancer, realizing early intervention treatment, and reducing the mortality of pancreatic cancer.

In the present invention, high-throughput sequencing is performed on normal samples and pancreatic cancer samples, and the 5-hydroxymethylcytosine (5-hmC) content in each gene is analyzed. An informative genetic marker that can be used to detect pancreatic cancer.

Therefore, the first aspect of the present invention relates to a genetic marker for detecting pancreatic cancer, including one or two or more of the following genes: sucrase-isomaltase (SI), type C exogenous lectin family 4 member C (CLEC4C), sorting protein 7 (SNX7), interstitial homeobox 2 (MEOX2), FAT atypical cadherin 1 (FAT1), flavin containing monooxygenase 3 (FMO3), cystic fibrosis transmembrane Conduction regulatory protein (CFTR), phospholipase 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 invention also relates to the use of the above-mentioned gene marker in detecting pancreatic cancer, and the content of 5-hydroxymethylcytosine in the pancreatic cancer gene marker is detected by high-throughput sequencing, thereby determining whether pancreatic cancer exists.

A second aspect of the present invention relates to a method for detecting pancreatic cancer, comprising the following steps: a) determining the 5-hmC content of the genetic marker according to the present invention in a normal sample and a subject sample; b) using normal The 5-hmC content of the gene marker in the sample is used as a control, and the 5-hmC content of the corresponding gene marker in the subject sample is standardized; c) the 5-hmC content of the standardized gene marker is performed Mathematically correlate and obtain a score; and 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 fluid, or is derived from whole genomic DNA in organelles, cells, and tissues. Among them, body fluids are blood, urine, sweat, sputum, stool, cerebrospinal fluid, ascites, pleural fluid, bile, pancreatic fluid, and the like.

In one embodiment, the 5-hmC content of the gene marker according to the present invention can be determined by any method known to those skilled in the art, such as, but not limited to, glucosylation, restriction Endonuclease 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), etc. The principle of glucosylation method is to use the T4 phage β-glucosyltransferase (β-GT) to transfer glucose to the hydroxyl position in the presence of glucose donor acceptor uridine diphosphate glucose (UDP-Glu), thereby Β-glucosyl-5-hydroxymethylcytosine (5-ghmC) is formed. At the same time, it can be quantified by isotopic labeling. Restriction enzyme method and chemical labeling method were further developed based on glucosylation method. The principle of the restriction enzyme method is that the glycosylation reaction changes the restriction characteristics of some restriction enzymes. 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) prevents cleavage. If the CpG site contains 5-hmC, then a band can be detected after glycosylation and enzymolysis, and there is no band in the unglycosylated control reaction; qPCR can also be used for quantitative analysis. In addition, other restriction enzymes may also prevent 5-ghmC digestion, which can be used for 5-hmC detection (such as: GmrSD, MspJI, PvuRts1I, TaqI, etc.). The principle of chemical labeling is: chemically modify the glucose on the substrate of the enzyme reaction into UDP-6-N3-glucose, transfer 6-N3-glucose to the methylol position, and generate N3-5ghmC. Subsequently, a molecule of biotin was added to each 5-hmC by a click chemistry method, and combined with the next-generation high-throughput DNA sequencing technology or single-molecule sequencing technology, the distribution of 5-hmC in genomic DNA could be analyzed. 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 quantitatively sequence 5-hmC with a single base resolution. First, 5-hmC is oxidized with KRuO4 to generate 5-formamidine cytosine (5fC). Sulphite sequencing. In this process, 5-hmC is first oxidized to 5fC, and then deaminated to form U. Usually, multiple detection methods are used for quantitative detection of 5-hmC.

In one embodiment of the invention, the 5-hmC content of the genetic markers of the invention is determined using chemical labeling methods in combination with high-throughput sequencing. In this specific embodiment, the method for determining the 5-hmC content of the genetic marker of the present invention includes the steps of: fragmenting DNA from samples from patients with pancreatic cancer and normal people; repairing the fragmented DNA ends The ends are complemented; the end-completed DNA is ligated with the sequencing adapter to obtain a ligation product; the 5-hydroxymethylcytosine in the ligation product is labeled by a labeling reaction; the 5-hydroxymethylcytosine-labeled DNA fragments to obtain enriched products; PCR amplification of the enriched products to obtain a sequencing library; high-throughput sequencing of the sequence library to obtain sequencing results; 5-hydroxymethylcytosine was determined based on the sequencing results Genetic content. Among them, the labeling reaction includes: i) covalently attaching a sugar with a modification group to a hydroxymethyl group of 5-hydroxymethylcytosine using glycosyltransferase, and ii) directly or indirectly linking biotin Click chemical substrates react with 5-hydroxymethylcytosine with modified groups. Among them, step i) and step ii) can be performed sequentially or simultaneously in one reaction. This labeling method reduces the number of samples required for sequencing, and the biotin tag on 5-hydroxymethylcytosine makes it show a higher kinetic signal in sequencing, which improves the accuracy of nucleotide recognition. In this embodiment, the glycosyltransferase includes, but is not limited to: T4 phage β-glucosyltransferase (β-GT), T4 phage α-glucosyltransferase (α-GT) and the same Or similarly active derivatives, analogs, or recombinases; the sugars with modified groups include, but are not limited to, sugars with azide modification (such as 6-N3-glucose) or other chemical modifications ( (Such as carbonyl, mercapto, hydroxyl, carboxyl, carbon-carbon double bond, carbon-carbon triple bond, disulfide bond, amine group, amido group, diene, etc.), of which the azide modification is preferred Carbohydrates; the chemical groups used to indirectly link biotin and click chemical substrates include, but are not limited to: carbonyl, thiol, hydroxyl, carboxyl, carbon-carbon double bonds, carbon-carbon triple bonds, disulfide bonds, amine groups , Amido, diene. In this embodiment, it is preferable to enrich the DNA fragment containing the 5-hmC tag by a solid phase material. Specifically, the 5-hydroxymethylcytosine-labeled DNA fragment can be bound to the 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 wafers with interface modification, such as artificial polymer beads (preferably 1nm-100um in diameter), magnetic beads (preferably 1nm-100um in diameter) , Agarose pellets, etc. (preferably 1nm-100um in diameter). The washing solution used for solid phase enrichment is a buffer solution 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 1mM to 1M), NaCl (0 -2M) or a buffer such as Tween20 (0.01% -5%). In this embodiment, it is preferable to perform PCR amplification directly on a solid phase to prepare a sequence library. If necessary, after performing PCR amplification on the solid phase, the amplified product can be recovered and then subjected to a second round of PCR amplification to prepare a sequence library. The second round of PCR amplification can be performed by 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 sequence library. Any set of purification reagents 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 spin column method, magnetic bead method, ethanol or isopropanol precipitation method, or a combination thereof. Optionally, prior to high-throughput sequencing, the quality of the sequence library is checked. For example, the sequence library is subjected to fragment size analysis and the concentration of the sequence library is absolutely quantified using the qPCR method. Quality-checked sequence libraries can be used for high-throughput sequencing. Then a certain number (1-96) of sequence libraries containing different barcodes are mixed at the same concentration and sequenced on the machine according to the standard method of the second-generation sequencer to obtain sequencing results. Various second-generation sequencing platforms and their related reagents known in the art can be used in the present invention.

In one embodiment of the present invention, it is preferable to compare the sequencing result with a standard human genome reference sequence, and select a sequence in which the gene marker is aligned, that is, select an alignment site and a gene characteristic (Such as histone modification sites, transcription factor binding sites, gene exon intron regions, gene promoters, etc.) The number of reads in the coincident region to represent the modification level of 5-hmC on the gene, so as to determine The content of 5-hmC on this gene marker. It is better to first clear the sequencing results of low-quality sequencing sites before performing the alignment. The factors that measure the quality of sequencing sites include, but are not limited to: base quality, reads quality, GC content, and repeat sequences. And overrepresented sequences. The various comparison software and analysis methods involved in this step are known in the art.

In one embodiment of the present invention, determining the 5-hmC content of a gene marker refers to determining the 5-hmC content of the gene marker over its entire length or determining the 5-hmC content of a fragment on the gene marker or a combination thereof. .

According to the present invention, after determining the 5-hmC content on each gene marker, the 5-hmC content of the gene marker in a normal sample is used as a control, and the 5-hmC content of the corresponding gene marker in the subject sample is used as a control. standardization. For example, if the 5-hmC content of the same gene marker in the normal sample and the subject sample is X and Y, respectively, the normalized 5-hmC content of the gene 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 according to the score. As used herein, "mathematical correlation" refers to any calculation method or machine learning method that correlates the 5-hmC content of a genetic marker from a biological sample with the diagnosis of pancreatic cancer. Those of ordinary skill in the art understand that different calculation 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 of mathematically correlating the standardized 5-hmC content of each gene marker and obtaining a score are as follows: multiply the standardized 5-hmC content of each gene marker by a weighting coefficient to obtain the gene The predictive factor t of the marker; add the predictive factors t of the markers of each gene to obtain the total predictive factor T; obtain the score P by mathematically transforming the total predictive factor T; if P> 0.5, the subject The subject's sample has pancreatic cancer; if P≤0.5, the subject's sample is normal. The weighting coefficients described herein refer to the factors that may affect the 5-hmC content (such as subject area, age, gender, below, smoking history, drinking history, family history, etc.), through the technology in the art Coefficients obtained by various advanced statistical analysis methods known to personnel.

The third aspect of the present invention also relates to a reagent set for detecting pancreatic cancer using the above-mentioned gene marker, which includes a reagent and an instruction for determining the 5-hmC content of the above-mentioned gene marker. Reagents for determining the 5-hmC content of genetic markers are known to those skilled in the art, such as T4 bacteriophage β-glucosyltransferase and isotope labeling (for glycosylation), restriction enzymes (for restriction Endonucleases), glycosyltransferases and biotin (for chemical labeling), reagents for PCR and sequencing, etc.

Compared with the prior art, the method for detecting pancreatic cancer according to the present invention is based on the 5-hmC content on the gene marker, so 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 asymptomatic people have a high acceptance of the test; (2) a wide range of DNA sources, and there is no blind detection zone in imaging; (3) High accuracy, high sensitivity and specificity for early pancreatic cancer, suitable for early screening of pancreatic cancer; (4) easy operation, good experience for the subject, and easy to perform recurrence and metastasis of pancreatic cancer Dynamic Monitoring. The gene marker of the present invention can be combined with other clinical indicators to provide more accurate judgments for screening, diagnosis, treatment and prognosis of pancreatic cancer.

The present invention is described in detail below with reference to the embodiments and the accompanying drawings, so that those skilled in the art can better understand the present invention and implement it.

Example 1. Screening of Pancreatic Cancer Gene Markers

1) Extraction of plasma DNA:

10 ng of plasma DNA was extracted from samples from 20 patients with pancreatic cancer and 20 normal subjects. This step can be performed using any method and reagents known to those skilled in the art that are suitable for extracting plasma DNA.

2) Complete the end of the plasma DNA, suspend A, and connect with the sequencing adapter:

Prepare 50uL of plasma DNA, 7uL of End Repair & A-Tailing Buffer and 3uL of End Repair & A-Tailing Enzyme according to Kapa Hyper Perp Kit instructions mix) reaction mixture (total volume is 60 uL), warmed at 20 ° C for 30 minutes, and then warmed at 65 ° C for 30 minutes. Configure the following ligation reaction mixture in a 1.5 mL low-absorption EP tube: 5uL Nuclease free water, 30uL Ligation Buffer, and 10 uL DNA Ligase. 5uL of sequencing adapter was added to the 45uL ligation reaction mixture, mixed, heated at 20 ° C for 20 minutes, and then maintained at 4 ° C. AmpureXP beads were used to purify the reaction product, and 20uL of a buffer containing Tris-HCl (10mM, pH = 8.0) and EDTA (0.1mM) was used to elute the final DNA ligation sample.

3) Mark 5-hydroxymethylcytosine:

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

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

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

First, prepare magnetic beads as follows: Take out 0.5uL C1 streptadvin beads (life technology) and add 100uL buffer (5mM Tris, pH = 7.5, 1M NaCl, 0.02% Tween20), vortex for 30 seconds, and then wash with 100uL (5mM Tris, pH = 7.5, 1M NaCl, 0.02% Tween20) washes the magnetic beads 3 times, and finally added 25 uL binding buffer (10mM Tris, pH = 7.5, 2M NaCl, 0.04% Tween20 or other surfactant), And mix well.

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

Finally, the magnetic beads were washed three times with 100 uL of washing solution (5mM Tris, pH = 7.5, 1M NaCl, 0.02% Tween20), the supernatant was removed by centrifugation, and 23.75uL of nuclease-free water was added.

5) PCR amplification:

Add 25uL of 2 X PCR master mix and 1.25uL PCR primer pair (total volume is 50uL) to the final system of the above steps, and perform amplification according to the temperature and conditions of the following PCR reaction cycle:

The amplified product was purified with AmpureXP beads to obtain the final sequence library.

6) High-throughput sequencing after quality inspection of the sequence library:

The obtained sequence library was subjected to concentration determination by qPCR, and the size and content of DNA fragments in the sequence library were determined using an Agilent 2100. The quality-checked sequence libraries were mixed at the same concentration and sequenced with Illumina Hiseq 4000.

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

The obtained sequencing results were subjected to preliminary quality control assessment. After the low-quality sequencing sites were removed, the reads that reached the sequencing quality standard were compared with the human standard genome reference sequence using the Bowtie2 tool. The featureCounts 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 the results of high-throughput sequencing, taking the factors that may affect the 5-hmC content as covariates, the weighting coefficients of each gene marker were obtained through logistic regression and elastic network regularization. The results are shown in Table 1.

Table 1: Mean standardized 5-hmC content and weighting coefficient of pancreatic oncogene markers of the present invention

As mentioned above, the average normalized 5-hmC content refers to the ratio of the average 5-hmC content of the gene marker in pancreatic cancer samples to the average 5-hmC content of the same gene marker in normal samples. As can be seen from Table 1, the 5-hmC content of the pancreatic cancer gene markers of the present invention is significantly different in normal samples and pancreatic cancer samples, and in addition to CLEC4C and PLPPR3, the 5-hmC content of other gene markers Significant increase compared to normal people.

Example 2. Effectiveness of Pancreatic Cancer Gene Markers

This example verifies 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 described in the present invention in the first batch of 82 samples (41 pancreatic cancer and 41 healthy controls) was determined according to the method of Example 1.

Multiply the normalized 5-hmC content of each gene marker by the corresponding weighting coefficient of the marker in Example 1 to obtain the prediction factor t of the gene marker, and then add the prediction factor t of each gene marker to obtain The total prediction factor T is obtained by converting the total prediction factor T through a mathematical logic operation (Logistic) according to the following formula:

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 differentiating the batch of samples according to the method of the present invention. As shown in FIG. 1, the method of the present invention can achieve a sensitivity of 95% and a specificity of 87%.

Finally, it should be noted that the above content is only used to explain the technical solution of the present invention, rather than limiting the protection scope of the present invention. Simple modifications or equivalent replacements made by those skilled in the art to the technical solution of the present invention do not depart from the present invention. The essence and scope of the technical solution of the invention.

no

FIG. 1 is a graph for distinguishing a pancreatic cancer sample from a healthy sample control according to the present invention.

no

Claims (10)

A genetic marker is used for detecting pancreatic cancer. The content of 5-hydroxymethylcytosine in a pancreatic cancer gene marker is detected by high-throughput sequencing, thereby determining whether pancreatic cancer exists. The genetic marker includes one or two More than the following genes: maltase (SI), type C exogenous lectin family 4 member C (CLEC4C), sorting protein 7 (SNX7), interstitial homeobox 2 (MEOX2), FAT atypical cadherin 1 (FAT1), flavin contains monooxygenase 3 (FMO3), cystic fibrosis transmembrane conductance regulating protein (CFTR), phospholipase phosphatase-related protein 3 (PLPPR3), alpha albumin (AFM), and collagen type V α2 chain (COL5A2). The use according to item 1 of the scope of the patent application, wherein the genetic markers include SI, CLEC4C, SNX7, MEOX2, FAT1, FMO3, CFTR, PLPPR3, AFM and COL5A2. A method for detecting pancreatic cancer, which comprises the following steps: a) determining the 5-hydroxymethylcytosine of the gene marker described in claims 1 and 2 of the patent application in normal samples and subject samples; Content; b) using the 5-hydroxymethylcytosine content of the gene marker in the normal sample as a control, normalizing the 5-hydroxymethylcytosine content of the corresponding gene marker in the subject sample, the normal sample and The 5-hydroxymethylcytosine content of the same gene marker in the subject sample is X and Y, respectively, then the normalized 5-hydroxymethylcytosine content of the gene marker in the subject sample is Y / X; c ) Mathematically correlate the 5-hydroxymethylcytosine content of the genetic marker normalized in step b) and obtain a score P; and d) obtain whether the sample of the subject has a disease according to the value of the score P There are test results for pancreatic cancer. The method according to item 3 of the scope of patent application, characterized in that step a) comprises the steps of: fragmenting DNA from samples of patients with pancreatic cancer and normal people; repairing and complementing the ends of the fragmented DNA Connect the end-completed DNA to the sequencing adapter to obtain a ligation product; label the 5-hydroxymethylcytosine in the ligation product by a labeling reaction; enrich the DNA fragment containing the 5-hydroxymethylcytosine label to obtain Enriched products; PCR amplification of the enriched products to obtain a sequence library; high-throughput sequencing of the sequence library to obtain sequencing results; the 5-hydroxymethylcytosine content in the gene was determined based on the sequencing results. The method according to item 4 of the scope of patent application, wherein the labeling reaction comprises: i) covalently linking a sugar bearing a modification group to a hydroxyl group of 5-hydroxymethylcytosine using a glycosyltransferase On the methyl group, and ii) react the click chemical substrate directly or indirectly with biotin and a 5-hydroxymethylcytosine with a modified group. The method according to item 3 or 4 or 5 of the scope of the patent application, wherein the step (a) is determining the content of 5-hydroxymethylcytosine on the full length of the gene marker or a fragment thereof. The method according to item 3, 4 or 5 of the scope of patent application, characterized in that step c) includes the step of: multiplying the normalized 5-hydroxymethylcytosine content of each gene marker by a weighting coefficient to obtain the gene marker The predictive factor t of the object; add the predictive factors t of each gene marker to obtain the total predictive factor T; obtain the score P by mathematically logically (Logistic) conversion of the total predictive factor T; if P> 0.5, the subject The subject sample has pancreatic cancer; if P≤0.5, the subject sample is normal. The method according to item 3 of the scope of the patent application, wherein the sample is a free DNA fragment from the body fluid of a normal person or a subject, or is derived from complete genomic DNA in organelles, cells, and tissues. The method according to item 8 of the scope of patent application, wherein the body fluid is blood, urine, sweat, sputum, stool, cerebrospinal fluid, ascites, pleural fluid, bile or pancreatic fluid. A reagent set for detecting pancreatic cancer, comprising: a) a reagent for determining the 5-hydroxymethylcytosine content of the gene marker described in the first patent application scope; and b) an instruction sheet; said The content of 5-hydroxymethylcytosine refers to the content of 5-hydroxymethylcytosine on the full length of the gene marker or a fragment thereof.