CN118957071A - Novel marker combination for multi-target pancreatic cancer detection and application thereof - Google Patents

Abstract

The invention provides a novel multi-target pancreatic cancer detection combined marker and application thereof, and a series of novel methylation sites which can efficiently distinguish pancreatic cancer, pancreatitis patients and healthy people are found from high-depth genome methylation (WGBS) data of pancreatic cancer tissues, beside-cancer tissues and pancreatitis patient samples, so that the novel multi-target pancreatic cancer detection combined marker can be used for efficiently detecting pancreatic cancer; the novel gene methylation locus is combined with other pancreatic cancer detection markers (such as CA 19-9), so that the AUC value can be further improved, and the sensitivity and the specificity of pancreatic cancer screening can be improved.

Description

Novel marker combination for multi-target pancreatic cancer detection and application thereof

Technical Field

The invention relates to the field of cancer screening, in particular to a novel marker combination for multi-target pancreatic cancer detection and application thereof.

Background

Pancreatic cancer (PANCREATIC CANCER) is a highly malignant cancer, called "cancer king". The five-year survival rate of pancreatic cancer is below 10% in most countries including China, and is far lower than that of colorectal cancer, esophageal cancer, liver cancer and other high-incidence malignant tumors. One of the reasons for the ultralow survival rate of pancreatic cancer is that early pancreatic cancer has no obvious clinical symptoms, and the existing clinic also has no high-performance marker for screening and diagnosing early and medium pancreatic cancer. Clinically common blood markers such as CA 19-9 and CEA have low sensitivity and specificity for pancreatic cancer screening diagnosis, so that most pancreatic cancer patients cannot be clearly diagnosed in early stages. Once pancreatic cancer has progressed to the middle and late stages, the likelihood that the patient will receive radical surgical resection therapy is greatly reduced, while clinically other effective non-surgical treatments for pancreatic cancer are very lacking (Diagnostics 2019; 9:18). Therefore, the development of high-performance tumor markers for screening pancreatic cancer, particularly early pancreatic cancer, is a great unmet clinical need and an important direction of efforts of the scientific and clinical oncology community.

CfDNA (cell free DNA) is a small fragment of DNA outside the cellular components of blood. cfDNA is theoretically thought to be released into the blood following apoptosis or necrosis, typically about 150 to 200 base pairs in length, with a half-life of about 0.5-1 hour (Nature REVIEWS GENETICS 2019; 20:71-88). In normal humans, cfDNA levels are minimal, whereas cfDNA levels are significantly elevated in tumor patients. In tumor patients, DNA released by tumors measured in peripheral blood is also known as circulating tumor DNA (ctDNA, circulating tumor DNA), which is part of cfDNA. In tumor patients, the proportion of ctDNA to cfDNA varies greatly, as little as less than 0.01%, and as much as more than 90%. For healthy asymptomatic population, if early diagnosis of tumor by liquid biopsy is successful, the 'tube blood cancer test' will become reality, which is clearly the most attractive place for liquid biopsy. Application discovery in this regard is currently underway (Annals of Oncology,2020,31 (6): 745-759; science,2020,369 (6499): eabb 9601).

CtDNA mutation is one possible direction for early screening and diagnosis of tumors. In a CANCERSEEK pan-cancer species screening study combining ctDNA mutation and protein detection in 2018, when the detection specificity was set at 99%, the sensitivity to detect different solid tumors was between 69-98%, with lung cancer being lowest and colorectal cancer (colorectal cancer, CRC) being highest, wherein the sensitivity to pancreatic cancer was 87% (Science, 2018,359 (6378):926-930). This finding demonstrates the exciting promise of early screening for cancer by ctDNA mutation detection. However, because of certain limitations of this study, such as the small number of samples of a single cancer species, most of the tumor cases incorporated are late in life rather than early, and more importantly, the results of the study are not validated in separate samples, so the effectiveness of the results in the real world requires more exploration.

Additional important markers in the genome include copy number variation (copy number variation, CNV) and methylation (methylation), both of which are early events in the development of cancer (HALLMARKS). Researchers reported that cfDNA methylation markers were used for screening malignant tumors such as liver cancer, intestinal cancer, breast cancer, etc. a paper published in Annals of Oncology at month (Nature materials 2017;16:1155-61;Clinical epigenetics 2018;10:1-10;Breast Cancer Research 2016;18:1-14).2020 analyzed the screening performance of >50 tumors using methylation protocols. When the investigator had set the specificity to 99%, the average detection sensitivity for the different carcinoma species was 43.9% in stage I-III tumors and 54.9% in stage I-IV tumors. The sensitivity was 63%,83%,75%, and 100% (Annals of Oncology,2020,31 (6): 745-759) in stages I, II, III, IV, respectively, for pancreatic cancer. Several limitations can be seen. For example, for phase III cases the performance is lower than phase II, which is likely due to randomness caused by the small number of samples. Although the performance for stage IV cases was 100%, the screening performance for stage I patients was only 63%. The clinical significance of early screening for pancreatic cancer in early cases is far higher than in late cases, because only in early cases, patients are likely to receive the opportunity for radical surgical resection. For patients with advanced findings, there is no clinically very effective means to greatly increase survival.

Therefore, there is a need to find a marker combination capable of efficiently distinguishing early pancreatic cancer from pancreatitis, thereby realizing high-sensitivity and high-specificity pancreatic cancer screening, promoting early detection and early treatment of pancreatic cancer, and meeting urgent clinical demands.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a novel multi-target pancreatic cancer detection combined marker, a kit and application thereof in pancreatic cancer detection. The novel series of methylation sites which can efficiently distinguish pancreatic cancer patients from pancreatitis patients are found from high-depth genome-wide methylation (WGBS) data of pancreatic cancer tissues, paracancestral tissues, chronic pancreatitis plasma, pancreatic cancer plasma and normal plasma samples, so that the kit can be used for efficiently detecting pancreatic cancer; the novel gene methylation locus is combined with other pancreatic cancer detection markers (such as CA 19-9, CEA and the like), so that the AUC value can be further improved, and the sensitivity and the specificity of pancreatic cancer screening can be improved.

The research group finds 9 methylation sites with obvious difference in methylation degree in pancreatic cancer patients and chronic pancreatitis patients, and further precisely screens out combinations of 6 methylation sites, so that the pancreatic cancer can be efficiently detected under the condition of less marker detection.

In one aspect, the present invention provides the use of a marker selected from any one or more of the nucleotide sequences set forth in Seq ID No.1 to Seq ID No.12 shown in Table 1 or a combination of the complete complementary sequences thereof for the preparation of a pancreatic cancer detection reagent.

TABLE 1 pancreatic cancer detection sites

The invention screens CpG-based methylation sites for distinguishing pancreatic cancer from non-pancreatic cancer by methylation sequencing of free DNA of plasma of patients with pancreatic cancer, chronic pancreatitis and normal human, and then further verifies by a large number of pancreatic cancer and non-pancreatic cancer plasma samples, and finally discovers and determines 6 target sequences with abnormal methylation in pancreatic cancer patients. Clinical verification shows that the 6 novel target sequences provided by the invention have obvious methylation degree differences in patients with pancreatic cancer and chronic pancreatitis.

The 6 target sequences are double-stranded DNA and have complementary sequences, and it is understood that the methylation site target sequences provided by the invention can be sense strands or antisense strands.

In some embodiments, the marker is a combination of 1,2, 3, 4,5, or 6 nucleotide sequences selected from the sequence list Seq ID No. 1-Seq ID No.12, or the complete complement thereof.

Further, the marker is a nucleotide sequence comprising any one or more of Seq ID No.1, seq ID No.2, seq ID No.3, seq ID No.9, seq ID No.11, and Seq ID No. 12.

Further, the marker is any 2, 3 or 6 nucleotide sequences of Seq ID No.1, seq ID No.2, seq ID No.3, seq ID No.9, seq ID No. 11, seq ID No.12 or a combination of the complete complements thereof.

The AUC values, detection sensitivities and specificities of the methylation sites corresponding to Seq ID No.1, seq ID No.2 and Seq ID No.12 are all significantly higher than those of the other methylation sites.

Further, the marker is a combination comprising the nucleotide sequence shown by Seq ID No.1 and Seq ID No.2 or the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.2, seq ID No.12 or the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.1, seq ID No.2 and the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.1, seq ID No.11 and the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.2, seq ID No.11 and the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.3, seq ID No.9 and the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.3, seq ID No.11 and the complete complement thereof, and the nucleotide sequence shown by Seq ID No.12, or a combination comprising the nucleotide sequence shown by Seq ID No.1, seq ID No.11 and the complete complement thereof.

Further, the marker is a combination comprising the nucleotide sequences shown by Seq ID No.1, seq ID No.2 and Seq ID No.12 or the complete complement thereof.

Through a large number of clinical pancreatic cancer and pancreatitis samples, the invention finds 3 hypermethylation sites which can particularly sensitively and specifically distinguish pancreatic cancer patients, pancreatitis patients and benign control individuals from the 6 target sequences, and the hypermethylation sites respectively have sequences shown as sequence tables Seq ID NO.1, seq ID NO.2 and Seq ID NO. 12. The data of the clinical plasma samples for detecting pancreatic cancer and pancreatitis show that the AUC value can reach 0.942 by only adopting the 3 methylation sites for pancreatic cancer detection, the sensitivity reaches 90 percent, and the specificity reaches 97 percent; the study proves that the pancreatic cancer diagnosis performance of the 3 methylation sites is not lower than or even slightly higher than the situation of 6 methylation site combinations, and the detection process is simpler, more convenient and efficient, and has the most application prospect.

Further, the markers also include a protein marker, which is the cancer antigen 19-9 (CA 19-9).

Due to technical limitations, the ability to detect cancer solely through methylation sites has also been a ceiling, and multiple sets of technologies have been developed that incorporate multiple categories of genomics, epigenetics, proteomics, and the like. Several clinical studies have shown that the sensitivity and specificity of the detection of multiple sets of chemical markers is superior to that of a single set of chemical markers. Therefore, on the basis of the methylation site provided by the invention, the detection of the level of the binding protein further improves the AUC value of pancreatic cancer detection and improves the detection sensitivity and specificity.

The level of the protein marker CA19-9 provided by the invention in serum of pancreatic cancer patients is obviously different from that of the protein marker in serum of pancreatitis patients.

The research proves that pancreatic cancer patients and non-cancer individuals can be distinguished more sensitively and specifically by detecting the methylation level of the series of novel methylation sites and combining the CA19-9 protein level in a serum sample. The data of the clinical pancreatic cancer and pancreatitis samples are tested, so that the combination provided by the invention can effectively distinguish patients with pancreatic cancer and non-cancer, the AUC value can reach 0.986 at most, the sensitivity reaches 93%, and the specificity reaches 97%.

In another aspect, the present invention provides a primer set for detecting pancreatic cancer, wherein the primer set is selected from any one or more of 12 sets of primers and probe sets shown in table 2.

TABLE 2 primer and probe combinations

In some embodiments, the primer combination is any 2,3, or 6 selected from the primers, probe combinations shown in table 2.

Further, the primer combinations were 3 sets of primer and probe combinations shown in Table 3.

TABLE 3.6 primer and probe combinations

Further, the primer combination also comprises a primer and a probe of an internal reference gene, wherein the internal reference gene COL2A1 has a sequence shown as a sequence table Seq ID No.13, a forward primer has a sequence shown as a sequence table Seq ID No.14, a reverse primer has a sequence shown as a sequence table Seq ID No.16, and a probe has a sequence shown as a sequence table Seq ID No. 15.

In yet another aspect, the invention provides a kit for detecting pancreatic cancer, the kit comprising any one of the primer and probe combinations described above.

Further, the kit or chip also comprises a reagent or material for detecting the protein, wherein the protein is CA19-9.

In yet another aspect, the present invention provides a marker combination for detecting pancreatic cancer, the marker combination comprising a methylation site and a protein marker, the methylation site being any one or more selected from the group consisting of Seq ID No.1 to Seq ID No.12 of the sequence listing; the protein marker is CA19-9.

In some embodiments, the methylation site in the marker combination is a combination comprising any 2,3, or 6 nucleotide sequences of Seq ID No.1, seq ID No.2, seq ID No.3, seq ID No.9, seq ID No. 11, seq ID No.12, or the complete complement thereof.

In some embodiments, the marker is a combination comprising the nucleotide sequence shown by Seq ID No.1 and Seq ID No.2 or the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.2, seq ID No.12 or the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.1, seq ID No.2 and the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.1, seq ID No.11 and the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.2, seq ID No.11 and the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.3, seq ID No.9 and Seq ID No.11, or the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.3, seq ID No.2, seq ID No.11 and the complete complement thereof, a combination comprising the nucleotide sequence shown by Seq ID No.3, seq ID No.11 and the complete complement thereof, a combination comprising the nucleotide sequence shown by Seq ID No.12, a combination comprising the nucleotide sequence shown by Seq ID No.3, seq ID No.11 and the complete complement thereof.

In some embodiments, the methylation site in the marker combination is a combination comprising the nucleotide sequences set forth in Seq ID No.1, seq ID No.2, and Seq ID No.12, or the complete complement thereof.

In yet another aspect, the present invention provides a marker combination for detecting pancreatic cancer, comprising a combination of the nucleotide sequences shown in Seq ID No.1, seq ID No.2 and Seq ID No.12 or the complete complement thereof.

The method for in vitro detection of pancreatic cancer through methylation sites provided by the invention comprises the following steps:

1) Separating genomic DNA or plasma free DNA in a biological sample to be detected;

2) Detecting the methylation state of the methylation site or combination of methylation sites;

3) And judging the state of the biological sample through the methylation site state of the target gene, and realizing in-vitro detection of pancreatic cancer.

The method for in vitro detection of pancreatic cancer by the methylation site and protein combination provided by the invention comprises the following steps:

1) Separating genomic DNA or plasma free DNA and serum in the biological sample to be detected;

2) Detecting the methylation state and serum CA19-9 protein level of the methylation site or combination of methylation sites;

3) And judging the state of the biological sample through the methylation site state of the target gene and the protein marker level, and realizing in-vitro detection of pancreatic cancer.

In some aspects, the method further comprises the steps of:

1) Separating serum and plasma of the biological sample to be detected, and extracting plasma free DNA of the biological sample to be detected;

2) Treating the DNA sample obtained in step 1) with a reagent to convert the 5-unmethylated cytosine base into uracil, the base after conversion to uracil being different from the 5-unmethylated cytosine in hybridization ability and being detectable;

3) Combining the DNA sample treated in step 2) with a polymerase chain reaction system comprising one or more of the following components: DNA polymerase, the primer or primer combination of the target sequence, the corresponding probe or probe combination, and a polymerase chain reaction buffer solution, and generating an amplification product after the polymerase chain reaction;

4) Detecting the amplified product with a fluorescent-labeled probe or a combination of probes, which can generate a fluorescent signal if combined with the amplified product; if the probe cannot bind to the amplification product, a fluorescent signal cannot be generated;

5) Determining the methylation status of at least one CpG of the target sequence of the target gene of interest based on whether a fluorescent signal is generated;

6) Measuring the concentration of CA19-9 in human serum by adopting a magnetic particle chemiluminescence immunoassay sandwich method;

in some embodiments, the polymerase chain reaction system wherein the DNA polymerase comprises a thermostable DNA polymerase, a hot-start DNA polymerase, or a polymerase lacking 5'-3' exonuclease activity.

The methylation status of at least one CpG in the target sequence of the target gene is determined by the difference between the cycle threshold Ct value of the PCR reaction or the Ct value of the target gene. Detection of the methylation state of one or more target sequences of a target gene of interest can be conveniently achieved by analyzing the methylation state of DNA in a biological sample using a PCR reaction.

In some embodiments, the conversion of unmethylated cytosine at the 5-position of the DNA to uracil preferably employs a reagent that is bisulfite.

Methylation modification of cytosine number 5 is a DNA modification mode widely existing in eukaryotic cell organisms, and methylation modification on DNA plays an important role in the growth and development of organisms and in the process of cell proto-canceration. Because of the same base-pairing properties as cytosine, 5-methylcytosine cannot be directly measured by means of one-generation sequencing or high-throughput sequencing. The most commonly used method for detecting 5-methylcytosine is to convert the DNA to be detected by bisulphite, and after alkaline hydrolysis, unmethylated cytosine is converted into uracil, while 5-methylcytosine is not converted. Uracil will pair complementarily to adenine when base paired, unlike cytosine paired complementarily to guanine, so that by detecting bisulfite treated DNA, the remaining unconverted cytosine can be determined by sequencing techniques, polymerase chain reaction techniques or DNA molecule hybridization-related techniques to determine which cytosine is methylated in the original DNA molecule. Therefore, the invention preferably adopts bisulphite as a methylation conversion reagent, and after the DNA sample to be detected is treated, the methylation state of the CpG dinucleotide sequence in the target sequence of the target gene is determined through relevant technologies such as sequencing, polymerase chain reaction or DNA molecular hybridization.

In some embodiments, the methods of the invention are applicable to analyzing samples in a mixed state, such as low concentrations of tumor cells present in blood, stool, or tissue. Thus, when analyzing the methylation status of CpG dinucleotide sequences in such samples, a person skilled in the art can use quantitative determination methods to determine the methylation level, such as percentage, ratio, fraction or degree, of CpG dinucleotide sequences, and the like, rather than the methylation state of a single nucleotide molecule. Accordingly, the methylation state described herein should be considered to include methylation modified states of single nucleotide molecules, including methylation states that are reacted by quantifying the level of methylation.

In some modes, the present invention employs a real-time fluorescent quantitative PCR mode to determine methylation status, such as: real-time fluorescent quantitative PCR using Taqman probes, real-time fluorescent quantitative PCR using fluorescent dyes, methylation-specific PCR (MSP) and the like are used to determine the methylation status of at least one CpG dinucleotide of a target sequence of a target gene. Because of the different base complementary pairing abilities of the gene target sequences of different methylation states, quantitative testing of methylation states in genomic DNA samples can be performed by real-time fluorescent quantitative PCR, where sequence discrimination occurs at the level of probe hybridization.

As a control, the COL2A1 gene was used in the present invention, and the genomic DNA after the reagent treatment was subjected to the test by designing the primer probe so as not to cover any CpG dinucleotide position.

Real-time fluorescent quantitative PCR can be used with any suitable probe, such as Taqman probes, MGB probes, scorpion probes, and the like. The fluorescent probe conventionally comprises a luminescent group, a nucleic acid sequence, a quenching group, and if necessary, some chemical modifications or special nucleotides such as a thioate nucleotide, a locked nucleic acid, etc.

In general, in real-time fluorescent quantitative PCR detection, the probe will be designed to have a melting temperature exceeding 10℃for the forward and reverse primers, which allows complete binding of the probe to the PCR product during annealing and extension. Typically, for example, a Taqman probe will hydrolyze during extension by a DNA polymerase having 5'-3' exonuclease activity, so that the fluorescent groups and quenching groups in the probe are far away, and resonance energy transfer between the fluorescent groups and quenching groups is destroyed, so that fluorescence emitted by the fluorescent groups can be detected by an instrument, and at the same time, as the PCR product increases gradually, the fluorescence signal will exhibit an exponential level rise over a certain period of time, and finally an "S" -shaped amplification curve is presented on a fluorescent quantitative PCR instrument.

Reagents for real-time fluorescent quantitative PCR include, but are not limited to: forward and reverse primers for target sequence of target gene, taqman fluorescent probe, optimized PCR buffer, deoxynucleotide triphosphate, DNA polymerase with 5'-3' exonuclease activity, etc.

The invention adopts but not limited to a magnetic particle chemiluminescence immunoassay sandwich method for detecting the target protein level, and other common detection methods such as a flow fluorescence luminescence method, an enzyme-linked immunosorbent method and the like.

In some embodiments, the presence or absence of a positive biological sample is determined by combining the methylation of the above sites or combinations of sites with the status of protein marker levels.

In a further aspect, the present invention provides the use of a marker for distinguishing pancreatic cancer from benign pancreatitis, said marker combination comprising a methylation site selected from any one or more of the sequences Seq ID No.1 to Seq ID No. 12.

In some embodiments, the methylation site in the marker combination is a combination comprising the nucleotide sequences set forth in Seq ID No.1, seq ID No.2, and Seq ID No.12, or the complete complement thereof.

Further, protein markers are included.

Further, the protein marker is CA19-9.

The methylation site for screening pancreatic cancer provided by the invention has the following beneficial effects:

1. Provides 12 brand new differential methylation sites, and the methylation state of the methylation sites in the plasma free DNA of the pancreatic cancer is obviously different from that of the plasma free DNA of the pancreatitis patient;

2. From the 12 methylation sites, optionally 2, 3 or 6 methylation sites in combination, can be used for high-sensitivity, high-specificity detection of pancreatic cancer;

3. From 12 methylation sites, pancreatic cancer detection AUC values of three methylation sites consisting of the Seq ID No.1, the Seq ID No.2 and the Seq ID No.12 are found, and the sensitivity and the specificity are good;

4. 3 methylation sites (Seq ID No.1, seq ID No.2 and Seq ID No. 12) are found to be combined with a protein marker CA19-9, so that the kit is used for noninvasive and rapid in-vitro detection of pancreatic cancer, pancreatic cancer and other pancreatic patients can be more effectively distinguished, the maximum AUC value can reach 0.986, the sensitivity can reach 93%, and the specificity can reach 97%;

5. The method is convenient and quick, and the detection result is highly consistent with the clinical gold standard detection result.

Drawings

FIG. 1 is a thermal diagram of sequencing data for pancreatic cancer and healthy human tissue samples WGBS in example 1, wherein tumor is a pancreatic cancer tissue sample; normal is a healthy human tissue sample;

FIG. 2 is a box plot of methylation level comparisons of 12 methylation sites in example 1 in different types of samples;

FIG. 3 is a schematic diagram showing the results of fluorescent quantitative PCR reaction for pancreatic cancer patient samples in example 2;

FIG. 4 is a schematic diagram of the detection result of the fluorescent quantitative PCR reaction of the healthy human sample in example 2;

FIG. 5 is a ROC curve obtained by normalizing the detection of 3 methylation sites in example 3;

FIG. 6 is a ROC curve obtained by normalizing the detection of the 3 methylation sites binding CA19-9 protein in example 3.

Detailed Description

The invention will be described in further detail below with reference to the drawings and examples, it being noted that the examples described below are intended to facilitate an understanding of the invention and are not intended to limit the invention in any way. The reagents used in this example are all known products and are obtained by purchasing commercially available products.

EXAMPLE 1 screening of methylation sites of genes

Starting from the actual clinical application of the product, 36 CpG sites are found and determined as qPCR candidate targets, and 12 target sequences with abnormal methylation in pancreatic cancer patients are screened out. The specific screening procedure for pancreatic cancer methylation qPCR candidate targets is as follows:

The screening process includes two stages, the first stage: screening a region which shows hypermethylation in pancreatic cancer tissues and demethylation in the paracancerous tissues (namely pancreatic cancer panel region) from WGBS data of 10 pairs of pancreatic cancer tissues and paracancerous tissue samples thereof and WGBS data of 20 normal human cfDNA samples (see figure 1), and designing and synthesizing corresponding capture probes; and a second stage: 254 cfDNA samples (including 116 pancreatic cancer cfDNA samples, 94 pancreatitis cfDNA samples, and 44 normal cfDNA samples) were captured and sequenced using probes, and by analyzing these data, 36 CpG sites that most distinguish pancreatic cancer cfDNA samples from non-pancreatic cancer cfDNA samples, with good complementarity and continuity, were screened out of the pancreatic cancer panel region as qPCR candidate targets.

Sequences were downloaded from NCBI database website according to region settings and converted to methylated sequences using METHYL PRIMER Express v1.0 software, i.e., "C" in the "CG" sequence was retained and all other "C" was converted to "T". As shown below. To ensure a well balanced GC content distribution in the design region sequence, the antisense strand is considered as a template for the sequence selection. Corresponding detection primers and probes were designed for the 36 hypermethylated regions according to the general primer design principle. Designed primers were screened using the following concentrations of template:

1) A pure positive template MJ is selected, the template is obtained by purifying after SSSI enzyme treatment, all CG sites have methylation modification, the template is a positive substance template verified by the optimal methylation test, and medium concentration and low concentration levels of 100 pg/mul and 10 pg/mul are selected.

2) Selecting a pure negative template WBC, wherein the template is taken from peripheral blood leukocyte gDNA of healthy people, contains the whole set of gDNA background, and better simulates the state of human cfDNA after ultrasonic disruption. 1 ng/. Mu.l was chosen as a simulated true high concentration sample.

3) A low abundance mixed template MJ/WBC, which is used to simulate the mixed state in a real sample, typically 5% as the state simulating a low abundance sample.

According to the design combination of primer screening, methylation detection systems are respectively prepared for comparison, and 100 pg/mu l and 10 pg/mu l of methylation positive templates, 1 ng/mu l of methylation negative templates and 5% abundance methylation mixed templates are subjected to comprehensive screening, wherein screening standards are shown in the following table 4. Finally, the primer combination is selected by taking the specificity, the detection rate and the Ct value of the amplification effect as judging indexes, and the screening result is shown in table 5.

TABLE 4 sequence combination screening criteria

TABLE 5 screening results

(UD means undetected)

After screening, the region 5 had a problem of nonspecific amplification, the region was excluded, and the regions 6 and 8 had a problem of insufficient amplification sensitivity, and the above 2 regions were also excluded. The remaining 9 regions all reached a better level in detection performance. Clinical performance verification of the target was then performed on clinical samples, using samples including 120 pancreatic cancer samples and 120 chronic pancreatitis samples. And (3) performing ROC analysis of a single region on the detection result, and removing the region with the AUC of less than 0.75 of the single region to remove the region with poor clinical performance, and reducing the number of the regions so as to meet qPCR detection, and simultaneously, ensuring that the better clinical performance is maintained when the subsequent region combination verification is performed.

TABLE 6 detection Performance of the 12 regions screened

After the above screening, 6 target sequences (having nucleotide sequences as shown in Seq ID No.1, seq ID No.2, seq ID No.3, seq ID No.9, seq ID No. 11, seq ID No.12, see Table 1 in the specification) hypermethylated in pancreatic cancer patients were finally found and determined. The box line graph of methylation level comparison of the 6 abnormal methylation sites in 3 different types of samples of pancreatitis plasma, pancreatic cancer plasma and healthy human plasma is shown in fig. 2, and the methylation level difference of the 6 methylation sites obtained by screening according to the invention for the different types of samples is obvious, so that pancreatic cancer, other pancreatic patients and normal people can be distinguished efficiently.

Example 2 detection of pancreatic cancer Using 2 methylation sites (or binding protein markers)

This example selects 2 of the 6 methylation sites screened in example 1 for pancreatic cancer detection. And the detection is carried out in two ways respectively: 1. pancreatic cancer is detected using 2 methylation sites; 2. pancreatic cancer was detected using a method of combining 2 methylation sites and protein markers.

1. Pancreatic cancer detection using 2 methylation sites

The specific method for detecting pancreatic cancer by using 2 methylation sites comprises the following steps:

Step 1, separating serum and plasma of a blood sample, extracting the plasma free DNA of the biological sample to be detected by using a magnetic bead method extraction reagent, wherein 30 cases are pancreatic cancer patients, and 30 cases are healthy people.

Step 2, methylation conversion treatment is carried out on the plasma free DNA sample extracted in the step 1 by using a methylation conversion reagent with bisulphite as a main component, and 5ng of plasma free DNA is added to convert unmethylated cytosine into uracil.

And 3, putting the converted plasma free DNA into a reaction system containing real-time fluorescence quantitative PCR for detecting the gene target sequence. Wherein the fluorescent probes for detecting 2 target sequences are respectively marked by FAM and ROX fluorescent dyes, and the fluorescent probes for detecting the internal reference gene COL2A1 are marked by VIC fluorescent dyes. Wherein, the upstream primer, the downstream primer and the probe refer to the upstream primer, the downstream primer and the probe corresponding to the 2 target sequences respectively as shown in Table 2.

The system for fluorescence quantitative PCR detection is a multiplex PCR system formed by mixing a plurality of target genes with primer probes of internal reference COL2A1, wherein the target genes are detected simultaneously with the internal reference COL2A 1. And (3) detecting at most 4 target genes and internal references simultaneously by a single tube, and detecting multiple tubes when the number of the target genes is more. In the reaction system, the forward and reverse primer input concentration of the target gene sequence is 0.167 mu M, the probe input concentration is 0.167 mu M, the real-time fluorescence quantitative PCR reaction system is 30 mu L, and the forward and reverse primer input concentration of the internal reference gene sequence is 0.083 mu M.

Step 4, a fluorescent quantitative PCR reaction detection procedure is set forth in table 7:

TABLE 7 multiplex PCR reaction procedure

And step 5, obtaining a fluorescent quantitative PCR reaction detection result.

In this example, the following groups of combinations comprising 3 methylation sites were used for the detection:

1、Seq ID NO.1、Seq ID NO.2

2、Seq ID NO.2、Seq ID NO.12

3、Seq ID NO.1、Seq ID NO.12

When two methylation sites corresponding to the nucleotide sequences shown in the SEQ ID NO.1 and the SEQ ID NO.2 in the 1 st group are selected, the detection results are shown in figures 3 and 4, wherein figure 3 shows the sample result of a pancreatic cancer patient, and all fluorescence signals of the methylation sites to be detected are detected to be positive; FIG. 4 shows a healthy human sample, wherein only the fluorescence signal of the control gene COL2A1 is detected, and the fluorescence signals of the rest sites to be detected are not detected, so that the sample is negative.

Analysis and judgment of the result of pancreatic cancer detection by using 2 methylation sites: 1) Recording the Ct value of each methylation site automatically output by software; 2) Respectively calculating the Ct value of each site in the sample and the internal reference COL2A1, and then carrying out normalization processing on the Ct: Δct (target sequence) = |ct (COL 2 A1) -Ct (target sequence) |; 3) M methylation sites, score for the ith methylation site is Mi. Mi is determined by a value of 0 or 1, respectively, based on the ΔCt (target sequence) value and the corresponding Youden's index. Let Mi=1 if ΔCt (target sequence) > Youden's index, and Mi=0 if ΔCt (target sequence) < Youden's index. Methylated M-score=sum_i++m (Mi). The test results are shown in Table 5.

The results obtained using the kit detection were corrected and calculated analytically according to logistic regression analysis. The threshold setting for M-score is set according to the ROC curve. And by synthesizing the detection results of the two target methylation sites, carrying out 10 times cross validation on the 60 samples, taking an average value to obtain a classified ROC curve, and calculating an AUC value, detection sensitivity and specificity.

The classification ROC curve was obtained by 10-fold cross-validation of these 60 samples by combining the detection results of methylation of 2 target sequences Seq ID No.1 and Seq ID No.2, see table 8, and taking the average.

TABLE 8.2 methylation level detection results for methylation sites

For the sample detection result, the pancreatic cancer detection level of the combination of the 2 methylation sites of the Seq ID No.1 and the Seq ID No.2 is adopted, the AUC=0.909, the sensitivity is 87%, the specificity is 95%, and the effect is obviously better than that of the combination of the methylation sites of other 2 groups. The results of the detection and analysis of groups 2 and 3 are detailed in the following examples 5 and Table 11.

2. Pancreatic cancer detection using a combination of 2 methylation sites and protein markers

In this example, 2 methylation sites of 1-6 groups were used to detect pancreatic cancer, respectively, in combination with a protein marker, wherein the protein marker was CA19-9.

The detection method of 2 methylation sites refers to the steps 1 to 5, and the detection of the protein marker CA19-9 also needs to be added with the step 6: taking the serum sample separated in the step 1, and measuring the concentration of the protein marker in the human serum by adopting a magnetic particle chemiluminescence immunoassay sandwich method.

The sandwich method process of the magnetic particle chemiluminescence immunoassay comprises the following steps: and mixing and incubating R1 (antibodies corresponding to CA19-9 are respectively adopted), a sample to be tested and M magnetic particles. Different sites of the protein marker in the sample are combined with the antibody coupled on the magnetic beads to form a solid-phase antibody-antigen complex; through cleaning, adding R2 reagent (respectively adopting CA19-9 corresponding secondary antibodies) for mixed incubation, and combining the complex with the labeled tumor marker antibody to form a solid-phase antibody-antigen-antibody sandwich complex; by washing, unbound antibodies and other substances are removed. Chemiluminescent substrates 1 and 2 are added to the reaction complex, and the chemiluminescent reaction is measured by relative luminescence intensities, which are proportional to the concentration of the tumor marker in the sample. The protein markers in the sample were subjected to a magnetic particle chemiluminescent immunoassay sandwich assay and the score was determined as P-value. The test results are shown in Table 9.

The analysis and judgment method comprises the following steps: in combination with the above analysis and judgment method for detecting pancreatic cancer by using 3 methylation sites alone, the following steps are also needed:

1) The CA19-9 detection value of each sample is normalized: p=log10p _CA19-9; p-score = sum a x P of the protein, where a = 3.8618;

2) The results obtained using the kit detection were corrected and calculated analytically according to logistic regression analysis. The threshold settings for M-score and P-score are set according to the ROC curve.

Detection performance is enhanced by integrating two complementary dimensions of methylation and protein markers. The integrated model was PC-score=m-score+p-score. In some embodiments, when the PC-score value is equal to or greater than a set threshold, the result indicates a positive detection of pancreatic cancer and/or early stage pancreatic cancer in the patient. In some embodiments, when the GC-score value is less than a threshold value, the result indicates a negative detection of pancreatic cancer and/or early stage pancreatic cancer in the patient.

3) The 60 samples were cross-validated by 10-fold by combining the 2 target methylation sites with the normalization of the protein CA19-9 detection results and averaged to obtain a classification ROC curve.

When 2 methylation sites corresponding to the nucleotide sequences shown in Seq ID No.1 and Seq ID No.2 of group 1 were selected, 10-fold cross-validation was performed on these 60 samples in combination with the normalization result after detection of protein CA19-9, and the average was taken to obtain a classification ROC curve as shown in FIG. 5, wherein the detection results of protein CA19-9 are shown in Table 9.

TABLE 9 detection of different sample protein levels

When the 2 methylation sites of the 1 st group are adopted and combined with the normalized result after the detection of the protein CA19-9, the pancreatic cancer detection effect can be further improved, the AUC=0.949, the sensitivity is 90%, and the specificity is 95%.

The normalization results after detection of the binding protein CA19-9 at the 2 methylation sites of groups 2 and 3 are detailed in the following example 5.

Example 3 detection of pancreatic cancer Using 3 methylation sites (or binding protein markers)

This example selects 3 out of the 6 methylation sites screened in example 1 for pancreatic cancer detection. And the detection is carried out in two ways respectively: 1. pancreatic cancer is detected using 3 methylation sites; 2. pancreatic cancer was detected using a method of combining 3 methylation sites and protein markers.

1. Pancreatic cancer detection using 3 methylation sites

In this example, the following groups of combinations comprising 3 methylation sites were used for the detection:

4、Seq ID NO.1、Seq ID NO.11、Seq ID NO.12

5、Seq ID NO.1、Seq ID NO.2、Seq ID NO.12

6、Seq ID NO.2、Seq ID NO.11、Seq ID NO.12

7、Seq ID NO.3、Seq ID NO.9、Seq ID NO.11

8、Seq ID NO.3、Seq ID NO.9、Seq ID NO.12

9、Seq ID NO.9、Seq ID NO.11、Seq ID NO.12

A specific method for detecting pancreatic cancer using 3 methylation sites comprises the steps of:

in the first step, 60 healthy people and pancreatic cancer patients are obtained, wherein the number of healthy people is 30, and the number of pancreatic cancer samples is 30. Serum and free plasma DNA of the extracted samples were separated.

In the second step, methylation reagent using bisulphite as main component is used to carry out methylation treatment on the plasma free DNA sample, and then plasma free DNA is added to convert unmethylated cytosine into uracil.

Third, referring to the upstream primer, the downstream primer and the probe corresponding to the 3 target sequences, respectively, as shown in Table 2, real-time fluorescent quantitative PCR detection was performed according to the method of example 2.

Aiming at the sample detection result of this time, the effect of adopting 3 methylation sites of the 5 th group is obviously better than the pancreatic cancer detection level of other groups. As can be obtained from the ROC curve, the auc=0.942 has a sensitivity of 90% and a specificity of 97% (fig. 5).

The results of the detection analysis of the 3 methylation sites of groups 4, 6-9 are detailed in the following example 5.

2. Pancreatic cancer detection using a combination of 3 methylation sites and protein markers

On the basis of the detection of pancreatic cancer by using 3 methylation sites, the number of the methylation sites is increased:

fourth, taking the serum sample separated in the first step, measuring the detection of the CA19-9 protein marker level in the human serum by adopting a magnetic particle chemiluminescence immunoassay sandwich method, and analyzing the result.

Fifth, the methylation of each gene and the value of the protein in the detected results were normalized, and by combining the delta Ct (target gene) results of 3 methylation sites and the detection results of the protein marker level, 10-fold cross-validation was performed on these 60 samples, and the average value was taken to obtain a classification ROC curve (fig. 6).

The normalization results after detection of binding proteins at 3 methylation sites of groups 4, 6-9 are detailed in example 5 below.

When 3 methylation sites of group 5 are adopted and the normalization result after CA19-9 protein detection is combined, the pancreatic cancer detection effect can be further improved, the AUC=0.986, the sensitivity is 93%, and the specificity is 97% (figure 6).

Example 4 detection of pancreatic cancer Using 6 methylation sites (or binding protein markers)

The 6 methylation sites screened from example 1 in this example were used for the detection of pancreatic cancer. And the detection is carried out in two ways respectively: 1. pancreatic cancer is detected using 6 methylation sites; 2. pancreatic cancer was detected using a method of combining 6 methylation sites and protein markers.

1. Pancreatic cancer detection using 6 methylation sites

The specific method for detecting pancreatic cancer by using 6 methylation sites comprises the following steps:

in the first step, 60 healthy people and pancreatic cancer patients are obtained, wherein the number of healthy people is 30, and the number of pancreatic cancer samples is 30. Serum and free plasma DNA of the extracted samples were separated.

In the second step, methylation reagent using bisulphite as main component is used to carry out methylation treatment on the plasma free DNA sample, and then plasma free DNA is added to convert unmethylated cytosine into uracil.

Third, referring to the 6 target sequences shown in Table 2, the upstream primer, downstream primer and probe were subjected to real-time fluorescent quantitative PCR detection in accordance with the method of example 2.

AUC for detection using 6 methylation sites reached 0.940, as described in example 5.

2. Pancreatic cancer detection using 6 methylation sites and protein marker combinations

A specific method for detecting pancreatic cancer by using a combination of 6 methylation sites and protein markers comprises the following steps: on the basis of the detection of pancreatic cancer by adopting 6 methylation sites, the number of the methylation sites is increased:

fourth, taking the serum sample separated in the first step, measuring the detection of the CA19-9 protein marker level in the human serum by adopting a magnetic particle chemiluminescence immunoassay sandwich method, and analyzing the result.

Fifth, the methylation of each gene and the value of the protein in the detected results are normalized, 10 times cross validation is performed on the 60 samples by combining the delta Ct (target gene) results of 6 target genes and the detection results of the protein targets, and an average value is taken to obtain a classified ROC curve, wherein AUC-methylation+protein=0.983, the sensitivity reaches 93%, the specificity reaches 96%, and the details are shown in example 5.

Example 5 Performance comparative analysis Using combinations of different methylation sites

Mathematical modeling analysis of different combinations of loci of relative cycle number Δct values of 6 methylation sites (Seq ID No.1, seq ID No.2, seq ID No.3, seq ID No.9, seq ID No. 11, seq ID No. 12) of 60 pancreatic cancer and healthy human samples obtained in example 1 was performed to investigate the use of 6 methylation sites and proteins as biomarker combinations for detecting pancreatic cancer.

First, we evaluated the performance of the above model of a single site of the 6 methylation sites for diagnosing pancreatic carcinogenesis, and calculated AUC values, respectively, and the results are shown in table 10.

TABLE 10 comparison of Performance of single methylation sites for pancreatic cancer diagnosis

As can be seen from Table 10, the 6 methylation sites provided in example 1 all have higher AUC values for diagnosing pancreatic cancer, and all have better diagnostic performance, especially the methylation sites corresponding to the nucleotide sequences shown in Seq ID No.1, seq ID No.2 and Seq ID No. 12.

Next, the diagnostic efficacy of the different combinations of methylation sites, or combinations of binding protein markers, listed in comparative examples 2-4 are shown in Table 11.

TABLE 11 comparison of models of methylation sites of different combinations for diagnosis of pancreatic carcinogenesis

As can be seen from tables 10 and 11, the diagnostic performance of the comparative multiple methylation site combination model using a single methylation site as the diagnostic model was lower, and the diagnostic performance of the multiple group of methylation binding protein markers was significantly better than that of the single group of markers using 2 methylation site combinations as the diagnostic model was lower than that of the 3 methylation site combination model.

When 2 methylation site combinations were chosen as diagnostic models, both AUC-methylation and AUC-methylation + protein were highest for group 1, and it was seen that when group 1 employed 2 methylation sites corresponding to the nucleotide sequences shown by Seq ID No.1, seq ID No.2, diagnostic performance was significantly higher than for the other 2 methylation site combinations.

When 3 methylation site combinations were selected as diagnostic models, the performance of the 3 methylation site combinations of groups 4-6 was top ranked, with both AUC-methylation and AUC-methylation + protein of group 5 being highest, it was seen that when 3 methylation sites corresponding to the nucleotide sequences shown in Seq ID No.1, seq ID No.2, and Seq ID No.12 were used in group 5, the diagnostic performance was significantly higher than the other 3 methylation site combinations.

When 6 methylation site combinations are selected as diagnostic models, the diagnostic performance is very close to that of the 3 methylation site combinations of group 5, so that the 3 methylation site combinations of group 5 are most preferably used.

The diagnostic properties of the different combinations of methylation sites preferred therefrom were further analyzed in this example, and the results are shown in Table 12.

TABLE 12 comparison of preferred multiple methylation site models for diagnosis of pancreatic carcinogenesis

As can be seen from table 12, the preferred combinations of several methylation sites and proteins in this embodiment can be used for high-efficiency detection of pancreatic cancer, wherein AUC can reach 0.986 at maximum, sensitivity reaches 93%, specificity reaches 97%, noninvasive, global and higher sensitivity and specificity pancreatic cancer screening are truly realized, and clinical requirements are completely met.

Example 6 detection of pancreatic cancer by different combinations of 3 methylation sites (or binding protein markers) in independent validation sets

The different 3 methylation site combinations and the combined proteins were performance verified in independent verification sets, which included 100 pancreatic cancer samples and 100 chronic pancreatitis samples, 100 pancreatic cancer samples and 150 healthy human samples.

(One) 100 pancreatic cancer samples and 100 chronic pancreatitis samples

Verification results the results are shown in table 13 below:

TABLE 13 comparison of pancreatic cancer Performance of combination diagnosis of different combination methylation sites in pancreatic cancer samples and chronic pancreatitis samples

(II) 100 pancreatic cancer samples and 150 healthy human samples

Verification results the results are shown in table 14 below:

TABLE 14 comparison of pancreatic cancer Performance of combination diagnosis of different combination methylation sites in pancreatic cancer samples and healthy human samples

As can be seen from tables 13 and 14, the detection performance can be effectively improved by methylation in combination with CA19-9, and the methylation markers of groups 1-3 can well distinguish pancreatic cancer patients from pancreatitis patients and pancreatic cancer patients from healthy people, wherein the combination of 1+2+12 has the optimal performance.

Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.

Sequence listing

Seq ID NO.1

Methylation site 1:

TAATTTATTTTTTTTGGATATTGGAGTTATTAATTGGCGTGTTTTTGTTTATTGGAGTATT

CGTATATTATTTTAAATTAAAATTATTAAGAGTTTTTTTCGCGTAGATTGTTGTTTTTTTA

GTTGTTTTCGATTTTGTTTTACGTTTGTCGGTTAGAGTTTTTCGGCGTTTTTTTCGTTTT

AGCGGAGTGCGTTGGGGCGCGTTAGGGTTAGGTTCGTCGGAGGAGCGCGTTTTTAGT

TTTTCGCGTATAGAGTCGTATTTCGSeq ID NO.2

Methylation site 2:

GTCGGTTGCGGTGGGGTCGGGTTGGAGGTCGCGGGTGAGGTTTGTGGTTAATTTCGC

GTTGTCGAGGTTTTATTTTTTCGAGTTTAGTTTGATTTTAGGTCGTTTTTAGGTCGGTG

TTTAGTTGAGGCGGGAACGTTGTAGTTTGGTTGAGCGTGATTTTTAGGTTTTGTGAGG

AAAAGTCGAGCGCGTTATATCGAGGCGTTAGTCGTTTATTTTATTATAAGGTAAAAGAT

TTATGTTGTTTTAGTTATTTTAAAGTTGGGAGATATATTGTATTTTTTATTAGATTTCGAA

TGTTTTTAGTGTSeq ID NO.3

methylation site 3:

AGAGTGGGGGCGTAGGGGGCGGGTTAGGTTTTTGGGCGCGGCGCGGGTTCGGGGGA

TTCGCGCGGTTGACGTTAGGTTATTTTTTAAATAGAGTCGGTAGCGCGTTTCGTTCGGT

ATTTTTCGAAGAGTTAGATCGCGGTCGGCGTTAGCGTTATCGTTCGGTTTATTCGTTAG

TTCGTATAGTCGCGTCGTCGTCGAGCGTTTCGTGAGCGGCGTTTCGAGGATTAGGAAT

GGGGTTTCGGGCGTTGGGCGCGTTTCGAATTCGGCGTACGTAAGAGTTTGGGAGCGT

TCGAGTCGTTCGGTTGTTCGGAGTTTTATCGTTTAGGATCGGGAGATGTTGGAAATGT

AATCGTTTGTTTTTCGAGGASeq ID NO.4

methylation site 4:

TCGAGTGTTGAAAAGGAGGGGCGTTTTTTTGGACGTAGGGGATATTTTTCGAGGGTTT

TTGTAGGTTTTTTGTAAGGATTAGGCGGTTTAGTTATTTTTCGTTTTGTATTCGTTTTCG

GTCGCGGCGTGTATTGAAGTTCGTTAGTGTTCGTTTTTGTAGCGAAGGTTTTTATAGTT

TAGGTTTTATAATTCGGGTAAAGGGGGAGTATTTGTGTGGGTATGGTTTTATAGTTTCG

TTTAGTATTAGTTTTAAATTTTTGTGSeq ID NO.5

methylation site 5:

ATAGTGGCGCGGTTGGGGCGGGCGGAGGAAGTGGGGGAGTTAAGGAGATATTTTAGC

GTTGGGATTCGGTAAGTTTTTTTTTTGAGTGGTTAGGGGGTTTCGTTTTTTTTTTCGAT

GTTTTTTGTTTTTTTTTGGGTTTTCGGAATTTAGTTTGTTTTAATCGTTTTCGTTGCGGG

TAGCGTTGGTTACGCGGTTTTCGTCGTCGGCGGTTTTTCGTGGTTAAGTATTTTTGGTT

TTGGAGTTTAGGGGTTGCGTTTTTTTTGGGGTCGGGGCGGGAGAGAGGATTTCGGTG

GTATTCGTTCGTGCGTTGGGCGSeq ID NO.6

Methylation site 6:

GTTGCGCGTTGATCGCGGGGTTCGATATGATGGTTGGTGGGTAGCGGGTCGCGCGGA

GGGTAGCGGCGAGGAAGCGTTTTCGGCGGGGTTCGGGTTTTGCGCGTTGGTTGGGGC

GTCGCGTTCGCGTTTTTGTAGTGTAGAGTTAGTCGTCGGAGGAGTTTTTAGGTTTTTT

GGTTTAGTTGAATGAATGGGGGAGGAAGGCGGGCGTCGGTTTTTTTTCGCGCGSeq ID NO.7

methylation site 7:

TGATTGCGGTTTTTTGGGGGATTTTGCGTTTAGTTCGAAAGTTGGTTTTGTTAAAAGA

CGTACGGGTAGGAAGGGCGAAAAGGAAATTTTGTATTTCGTCGCGTTGGGTTTTTCG

AGTTCGTGCGTAAAGCGGTTTACGAGTTTTGGTTTCGTATTTGTAGAGGATAAGAGTT

AATGTTTAAAAAAGAATAGCGGAGGAATCGGTTGGCGCGGTTAGTTGGAACGTTGGA

TCGTAGTGCGTTTAGGGAAGGTCGGGGGCGTTCGTCGGTTTTAGTTTTTAGTGGTTTT

TTTTTACGTCGGTTCGCGCGTGTTTTTGTTTATAAGATTTGGGGCGTTTTGGTTAGATT

TGGATGGTAGGTTTTTTCGTTATTTTCGGTTCGGTTTCGGGGGCGTGGTTTGGCGCGG

GGGCGGGTTTAAGTTATCGCGGGTGTTTGATTATTTTGATAGGTTTTTAAATTTTTTTTA

GTCGTTTGGCGTTCGCGGTGCGTTTTAGAGTTTTAGGGTGGTACGCGGCGGCGTTTTT

TTAGATTTAGAGGCGTTTTTGTCGATTTTTACGCGGTTTCGGGTTTTTTTTTTTTTTTAA

ATTTTCGTTTTATTCGTTAAGTTTCGGTTTTTAGTTTTAGATATTCGTATCGGCGGTTTTT

TTTCGTTTTCGGAGTTTTTGGTTTTGGAGTTTTTATTATTTTTTTTTTTTTTCGTTTTTTT

TGATTTTTTTAGTTTCGAGTTAAATGGATTTTAAAGAACGAAATAAAGGGSeq ID NO.8

methylation site 8:

TTTTAGATTAGGGTGTTTAGGGGTGGTTATTTGGAGGAGGTGATATTTGAGACGTTAA

CGAGAACGAGTTAGTTTAGTTTAGAGGAGGTTTAGGGGAAACGCGGTTTAGGTTTGA

GATTATAAAGGGTATATTAGTGGTTACGAAGTTTTTTGGGGGGAAAATTCGGTAGGTC

GTCGTCGTCGTTTCGTTAGTAGTTATCGATTTTCGTTTTGAGAGTTCGGAGTTGACGC

GAAGGAGGGGGTTCGTTTTGGCGCGTTTTTTTTTTCGCGTTATGTATTAGTTAGATGGG

GAGTCGGGGTGGTTAGGTTTTGGGTCGGGTAAGTTGGATTGTTCGGTCGATTTGTTTT

TATTTTTCGTTTTTTTTTTTTCGGGGCGGTCGAAGAGTATCGGGTTGATTTTTTTTTTAA

GTGAAATTGSeq ID NO.9

methylation site 9:

GCGCGGTAGCGTGTTACGGACGGGGACGAGGGGATGGCGGGGACGACGACGTGGGC

GGTGGGAGTAGAGTGTTTGCGATTTTCGGGGTAGGAAGGAGGGGCGGGATGGTTTTT

CGCGTTCGAAATTTACGGCGTGGCGGACGCGGAGGAGTTTCGAGTTTTATAATTAAGG

GGTGGGAAGGAAAAGGGATAGAGCGAGATAGAGCGTTTTCGAGAAASeq ID NO.10

methylation site 10:

ATATTTGGTTTTAAGGTTATTATTTGATATAAAAATATAATGTTGGTTTTTTAAATAGGTA

ATTTATATTGATATTTATATAGTTTTTATTTATATTTGCGATTATAAGGTAGTATTTTCGGG

TTTTCGTCGGTTACGATTCGTTTTTAGAGAGTATTCGCGTATAATTTTATTTTATTTACGA

TTTTGATTTTTTTTTTTTTATTTTTTTAATTTTATTCGGGACGAGATTATTAGGTTTTATTT

TTATTTTTATTTTTATSeq ID NO.11

methylation site 11:

GGGGAAAGGGTCGGGTAAGGTTCGGATGGAGTAGTTTCGGGAAGGTGCGGGTTTCG

GCGATCGTTGGATGGTTGTCGTTTTTTCGACGAGTGCGTTTTTCGTAAATGTTTTTTGG

GGTAAGGGTTTTCGGTTATTGAGTTATGTATAGTAAGTTTAGATCGTAAAAAGATTCGC

GAAGGGTAGAATATGTCGGTTTTTTCGGTTCGTCGTCGTTTTTAGTTTAGGATGTTTTC

GAATTTTGGAGTCGAATAGGTTGGGTTTTTTTAGCGCGTTTTATCGCGGTTTAGTTTTA

TTATTTCGCGCGTTGTTCGTTTTTTCGCGTTTTTGGATTCGTSeq ID NO.12

methylation site 12:

TTATTATCGCGGAATTTTGAAAGCGTAGATTTGTTTTGAAGTTTTGAGCGATTTTTCGT

ATTAGGCGTTGGAAAGGTAATTTGCGGATTAGTAGTTTAGTCGATTTTTGATAGCGGCG

AATTTCGCGTAGAGTTACGGTGTCGGGACGGTAGCGATGAGGTTTTTTTACGTCGTCG

GTGGTCGGCGCGTTTTTTCGAGTCGTTTTAGTGGTTAGTTGTTTGCGCGGCGTTTTGTT

ATCGGTG

Seq ID NO.13

COL2A1：

TTTTTTGTAAGGAGGGATGTGGAGGGATAGAGGAGTAGTAGGTAAGGTTAGTAGGAG

GTGATATAGGTAGGGAGGATTAGGTTAAGGTTGGGAGGAGTTTATATTTGGTGTT Seq ID NO.14

forward primer of COL2 A1:

ATGTGGAGGGATAGAGGAGTA Seq ID NO.15

Probes for COL2 A1:

CACCTCCTACTAACCTTACC Seq ID NO.16

reverse primer of COL2 A1:

CCTCCCAACCTTAACCTAAT Seq ID NO.17

Forward primer for methylation site 1:

GTTGTTTTCGATTTTGTTTTACG Seq ID NO.18

probe of methylation site 1:

CGCCGAAAAACTCTAACCGA Seq ID NO.19

Reverse primer of methylation site 1:

CGCACTCCGCTAAAACG Seq ID NO.20

Forward primer for methylation site 2:

TGATTTTTAGGTTTTGTGAGGAAA Seq ID NO.21

probe of methylation site 2:

CTCGATATAACGCGCTCGA Seq ID NO.22

Reverse primer of methylation site 2: AATCTTTTACCTTATAATAAAATAAACG SEQ ID NO.23

Forward primer for methylation site 3: GTTTCGTTCGGTATTTTTCGAA SEQ ID NO.24

Probe of methylation site 3: TAACGCCGACCGCGATCT SEQ ID NO.25

Reverse primer for methylation site 3: CGAATAAACCGAACGATAACG SEQ ID NO.26

Forward primer for methylation site 4: GGATTAGGCGGTTTAGTTATTTTTC SEQ ID NO.27

Probe of methylation site 4: CGTTTTCGGTCGCGGC SEQ ID NO.28

Reverse primer of methylation site 4: AACGAACACTAACGAACTTCAATACA SEQ ID NO.29

Forward primer for methylation site 5: CGTTGCGGGTAGCGTTG SEQ ID NO.30

Probe of methylation site 5: TTTTCGTCGTCGGCGGT SEQ ID NO.31

Reverse primer of methylation site 5: AACTCCAAAACCAAAAATACTTAACC SEQ ID NO.32

Forward primer for methylation site 6: GGGGTTCGGGTTTTGC SEQ ID NO.33

Probe of methylation site 6: CGTCGCGTTCGCGTTT SEQ ID NO.34

Reverse primer of methylation site 6: CCGACGACTAACTCTACACTACAA SEQ ID NO.35

Forward primer for methylation site 7: GTTCGTCGGTTTTAGTTTTTAGTG SEQ ID NO.36

Probe of methylation site 7: CGCGCGAACCGACGTA SEQ ID NO.37

Reverse primer for methylation site 7: CGCCCCAAATCTTATAAACAAAA SEQ ID NO.38

Forward primer for methylation site 8: GGGGGGAAAATTCGGTAGG SEQ ID NO.39

Probe of methylation site 8: CTAACGAAACGACGACGACG SEQ ID NO.40

Reverse primer of methylation site 8: ACTCTCAAAACGAAAATCGATAACT SEQ ID NO.41

Forward primer for methylation site 9: ATGGTTTTTCGCGTTCGAA SEQ ID NO.42

Probe of methylation site 9: TTTACGGCGTGGCGGAC SEQ ID NO.43

Reverse primer of methylation site 9: CCCCTTAATTATAAAACTCGAAACTC SEQ ID NO.44

Forward primer for methylation site 10: TATATTTGCGATTATAAGGTAGTATTTTC SEQ ID NO.45

Probe of methylation site 10: TTTCGTCGGTTACGATTCGT SEQ ID NO.46

Reverse primer of methylation site 10: TAAAATTATACGCGAATACTCTCTAAA SEQ ID NO.47

Forward primer for methylation site 11: ATTCGCGAAGGGTAGAATATGTC SEQ ID NO.48

Probe of methylation site 11: TTCGGTTCGTCGTCGTTT SEQ ID NO.49

Reverse primer of methylation site 11: CAAAATTCGAAAACATCCTAAACTA SEQ ID NO.50

Forward primer for methylation site 12: GCGGATTAGTAGTTTAGTCGATT SEQ ID NO.51

Probe of methylation site 12: TACGCGAAATTCGCCGC SEQ ID NO.52

Reverse primer of methylation site 12: CGTCCCGACACCGTAA.

Claims (10)

1. Use of a marker for preparing a pancreatic cancer detection reagent, characterized in that the marker is a combination of nucleotide sequences shown in any one or more of Seq ID No.1 to Seq ID No.12 or complete complements thereof.

2. The use according to claim 1, wherein the marker is a nucleotide sequence comprising any one or more of Seq ID No.1, seq ID No.2, seq ID No.3, seq ID No.9, seq ID No. 11, seq ID No. 12.

3. The use according to claim 2, wherein the marker is a combination comprising any 2, 3 or 6 nucleotide sequences of Seq ID No.1, seq ID No.2, seq ID No.3, seq ID No.9, seq ID No. 11, seq ID No.12 or the complete complement thereof.

4. The use according to claim 3, wherein the marker is a combination comprising the nucleotide sequence shown by Seq ID No.1 and Seq ID No.2 or the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.2, seq ID No.12 or the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.1, seq ID No.2 and Seq ID No.12, or a combination comprising the nucleotide sequence shown by Seq ID No.1, seq ID No.11 and the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.2, seq ID No.11 and the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.3, seq ID No.9 and Seq ID No.11, or the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.3, seq ID No.11 and the complete complement thereof, a combination comprising the nucleotide sequence shown by Seq ID No.11 and the complete complement thereof, or a combination comprising the nucleotide sequence shown by Seq ID No.12, a combination of the nucleotide sequence shown by Seq ID No.1, seq ID No.11 and the complete complement thereof.

5. The use according to claim 4, wherein the marker is a combination comprising the nucleotide sequences shown in Seq ID No.1, seq ID No.2 and Seq ID No.12 or the complete complement thereof.

6. The use according to any one of claims 1 to 5, wherein the marker further comprises a protein marker consisting of CA 19-9.

7. A primer combination for detecting pancreatic cancer, wherein the primer combination is any one or more groups selected from 12 groups of primers and probe combinations shown in the following table:

8. A kit for detecting pancreatic cancer, comprising the primer and probe combination of claim 7.

9. A marker combination for detecting pancreatic cancer, comprising a methylation site and a protein marker, wherein the methylation site is any one or more selected from the group consisting of Seq ID No.1 to Seq ID No.12 in the sequence listing.

10. The marker combination according to claim 9, wherein the methylation site comprises a combination of the nucleotide sequences shown in Seq ID No.1, seq ID No.2 and Seq ID No.12 or the complete complement thereof.