CN114908166A - A group of specific methylation markers associated with colorectal cancer and their detection kits - Google Patents

Abstract

The invention discloses a group of colorectal cancer-related specific methylation markers and a detection kit thereof. The methylation markers are 15 target CpG sites located on B3GALNT1, C6orf97, FAM72A, FAM72B, LIFR, OSMR, ZNF264 and ZNF543 genes. The 15 CpG sites showed a specific hypermethylation state in colorectal cancer tissues compared with other types of cancer tissues. The present invention detects the methylation level of 10 CpG sites in colorectal cancer plasma cfDNA for the first time. By optimizing primers, probes and reaction systems, three multiplex digital PCR detection systems are established. The detection sensitivity of the detection method is It is obviously better than the conventional MethyLight detection method, and the detection limit can reach 0.032%. Multiplex digital PCR was used to characterize and quantify the methylation levels of the seven amplified fragments by calculating the number of methylated molecule copies per milliliter of plasma. Certain ability to identify colorectal adenomas and differentiate patients with other tumors.

Description

A group of specific methylation markers associated with colorectal cancer and their detection kits

technical field

The invention relates to a group of colorectal cancer-related methylation markers, and also relates to a kit for diagnosing colorectal cancer in a population, and belongs to the technical field of biomedicine.

Background technique

According to GLOBOCAN 2020 estimates, colorectal cancer (CRC) has the highest morbidity and mortality worldwide. The onset of colorectal cancer is insidious, and more than half of the patients are in the middle and late stages at the time of diagnosis. Therefore, early screening and effective removal of colorectal cancer or precancerous lesions can greatly curb the occurrence and development of cancer and improve the survival rate of patients. Colonoscopy is currently a routine clinical method for colorectal cancer screening and endoscopic treatment, but its invasiveness and relatively complex bowel preparation process makes the compliance of patients not high, only 30-55%. Some non-invasive screening methods, such as fecal occult blood test, serum tumor markers CEA and CA199, all have limitations such as high false positive rate or limited sensitivity.

In recent years, liquid biopsy has emerged in the field of new tumor marker research and development, that is, it can identify molecules released by the lysis or apoptosis of tumor primary or metastatic cells into blood, urine, saliva, pleural effusion and other body fluids, such as circulating Tumor cells (CTC), circulating tumor DNA (ctDNA), circulating tumor RNA (ctRNA) and exosomes, etc. Compared with traditional surgical biopsy, liquid biopsy has lower invasiveness and heterogeneity, and allows flexible sampling for dynamic monitoring of tumors (treatment effect and recurrence, etc.). The minimally invasive nature of blood testing and its convenience in routine medical examinations make it the most commonly used biological sample for liquid biopsies. In tumor patients, tumor-specific genetic and epigenetic changes such as mutation, copy number variation, and methylation level are used to identify ctDNA in cfDNA, thereby realizing non-invasive diagnosis of cancer patients. DNA methylation is a dynamically reversible and stably inherited epigenetic change that occurs on CpG dinucleotide cytosines. DNA methylation is widespread in the genome and is critical for gene regulation, transcript silencing, and gene imprinting. Studies have shown that abnormal DNA methylation occurs at an early stage in a variety of cancers, including colorectal cancer, and is cell- or tissue-specific, making it possible to construct methylation profiles of different cancer tissues or cells. Therefore, the combination of epigenetic modification of DNA methylation and blood testing makes cfDNA methylation detection the most promising biomarker for liquid biopsy. SEPT9 Gene Methylation, the first commercially available blood cfDNA methylation marker for colorectal cancer screening, has been extensively tested and reviewed, and results show a sensitivity range of 37% for the diagnosis of colorectal cancer between 79% and 99%, and specificity between 79% and 99%. However, studies have suggested that SEPT9 has different degrees of elevated methylation levels in cfDNA of patients including head and neck squamous cell carcinoma, lung cancer and liver cancer, and the sensitivity in liver cancer is as high as 82.7%. Therefore, the early non-invasive detection of colorectal cancer should take into account the tumor specificity while improving the sensitivity, and further exploration of colorectal cancer-specific cfDNA methylation diagnostic markers is still needed.

SUMMARY OF THE INVENTION

In order to fill the gap that there is currently no methylation marker that can specifically detect colorectal cancer, one of the objectives of the present invention is to provide a set of methylation markers that can be used to specifically detect colorectal cancer.

The second purpose of the present invention is to provide a kit for detecting colorectal cancer based on the above methylation markers.

In order to achieve the above object, the present invention has adopted the following technical means:

The present invention firstly proposes a group of specific methylation markers related to colorectal cancer, the methylation markers are B3GALNT1, C6orf97, FAM72A, FAM72B, LIFR containing at least one CpG methylation site, OSMR, ZNF264 or ZNF543 gene sequences or fragments thereof, or any combination of said gene sequences or fragments thereof;

According to the genome annotation information of the 450K chip (GRCh37/hg19), there is one CpG site on the B3GALNT1 gene, namely cg20841906, which is located at the 160822911 nucleotide of the reverse strand of chromosome 3;

Among them, there is one CpG site on the C6orf97 gene, namely cg00117463, which is located at the 151815241 nucleotide of the positive strand of chromosome 6;

Among them, there are two CpG sites on the FAM72A gene, cg00552973 and cg10439914, respectively, cg00552973 is located at nucleotide 206137054 of the reverse strand of chromosome 1, and cg10439914 is located at nucleotide 206137759 of the positive strand of chromosome 1;

Among them, there are 6 CpG sites on the FAM72B gene, which are cg07784526, cg18948743, cg08728856, cg00198436, cg07236150 and cg09169215. At 120838323 nucleotides of the At 120839385 nucleotides, cg09169215 is located at 120839390 nucleotides of the anti-strand of chromosome 1;

Among them, there is one CpG site on the LIFR gene, namely cg18174928, which is located at nucleotide 38557085 of the positive strand of chromosome 5;

Among them, there is one CpG site on the OSMR gene, namely cg17528648, which is located at the 38846100th nucleotide of the positive strand of chromosome 5;

There are 2 CpG sites in ZNF264 gene, cg26970847 and cg11140785 respectively, cg26970847 is located at the 57702793 nucleotide of the reverse strand of chromosome 19, and cg11140785 is located at the 57703301 nucleotide of the reverse strand of chromosome 19;

There is one CpG site in ZNF543 gene, namely cg14786398, which is located at the 57831816 nucleotide of the reverse strand of chromosome 19.

Further, the present invention also provides a digital PCR kit for colorectal cancer-related gene methylation detection, the kit includes FAM72A, FAM72B, LIFR, OSMR, ZNF264 and ZNF543 for colorectal cancer-related genes. The combination of digital PCR primers and probes for the detection of gene CpG methylation sites, the kit also includes primer pairs and probes required for the amplification fragment of the internal reference gene ACTB gene that needs to be corrected for the loading amount, the primers and The probe sequences are as follows:

Among them, the FAM72A primer pair is used to amplify the FAM72A gene fragment, and the probe is used to detect a CpG site existing on the gene fragment, namely cg00552973, which is located at the 206137054 nucleotide of the reverse strand of chromosome 1; FAM72B_1 primer For the amplification of the FAM72B gene fragment, the probe is used to detect the two CpG sites present on the gene fragment, cg07784526 and cg18948743, respectively. At the 120838323 nucleotide of the positive strand of chromosome 1; the FAM72B_2 primer pair is used to amplify the FAM72B gene fragment, and the probe is used to detect the three CpG sites present on the gene fragment, which are cg00198436, cg07236150 and cg09169215, cg00198436 is located at nucleotide 120839380 of the anti-strand of chromosome 1, cg07236150 is located at nucleotide 120839385 of the anti-strand of chromosome 1, and cg09169215 is located at nucleotide 120839390 of the anti-strand of chromosome 1; LIFR primers For the amplification of the LIFR gene fragment, the probe is used to detect a CpG site present on the gene fragment, namely cg18174928, which is located at nucleotide 38557085 of the positive strand of chromosome 5; the OSMR primer pair is used for amplification. The OSMR gene fragment was added, and the probe was used to detect a CpG site existing on the gene fragment, namely cg17528648, which was located at the 38846100th nucleotide of the positive strand of chromosome 5; ZNF264 primer pair was used to amplify the ZNF264 gene fragment , the probe is used to detect a CpG site existing on the gene fragment, namely cg11140785, which is located at the 57703301 nucleotide of the reverse strand of chromosome 19; the ZNF543 primer pair is used to amplify the ZNF543 gene fragment, and the probe is used One CpG site in the detected gene fragment, namely cg14786398, is located at nucleotide 57831816 of the reverse strand of chromosome 19.

Wherein, preferably, the 5' end of the probe contains a fluorescent reporter group, and the fluorescent reporter group includes any one of FAM, VIC/HEX, NED, ROX, TET, JOE, TAMRA, CY3, CY5 The methylation probe and the internal reference gene probe used for the detection of CpG methylation sites use different fluorophores, the 3' end of the probe contains a fluorescence quenching group, and the fluorescence quenching The quenching group includes any one of MGB, BHQ-1, BHQ-2, and BHQ-3.

Wherein, preferably, combining 7 amplified fragments into three multiplex digital PCR detection systems, multiplex ddPCR system 1 is used to amplify LIFR, ZNF264 and ZNF543 fragments, multiplex ddPCR system 2 is used to amplify FAM72A and FAM72B_2 fragments, multiplex ddPCR system System 3 is used to amplify FAM72B_1 and OSMR fragments; the digital PCR reaction system and conditions of the kit are as follows:

Digital PCR reaction system: PCR master mix 2×ddPCR Supermix forProbes 10μL, primer final concentration range between 0.2μM-0.9μM, probe final concentration between 0.1μM-0.5μM, 0.002-70ng/uL template DNA, Make up RNase-free water to 21uL;

The digital PCR reaction conditions are as follows:

Pre-denaturation at 95°C for 10 minutes, denaturation at 94°C for 30 seconds, annealing at 60°C for 60 seconds, for a total of 40 cycles, inactivate the enzyme at 98°C for 10 minutes, and then keep at 4°C;

Wherein, preferably, the template DNA is DNA in the blood of the test subject.

Wherein, preferably, the template DNA is circulating cell free DNA (cfDNA), circulating tumor DNA (ctDNA) or circulating tumor cells (circulating tumor cells, CTCs) DNA in peripheral blood plasma or serum.

Further, the present invention also proposes the use of the digital PCR kit in the preparation of colorectal cancer diagnostic reagents.

Compared with the prior art, the beneficial effects of the present invention are:

The present invention utilizes TCGA and other databases to analyze the tissue-specific methylation profile of colorectal cancer, and screen out the tumor methylation-specific markers, and mainly screen out genes distributed in B3GALNT1, C6orf97, FAM72A, FAM72B, LIFR, OSMR, ZNF264 and ZNF543 15 CpG sites on it. The 15 CpG sites showed colorectal cancer tissue-specific hypermethylation status in 29 types of cancer tissues including colorectal cancer and peripheral blood leukocytes. Compared with the existing FDA-approved SEPT9 methylation markers, the 15 CpG sites screened by the present invention have higher colorectal cancer specificity. In addition, the present invention also proposes for the first time primers, probes and kits for detecting methylation of 10 target CpG sites on FAM72A, FAM72B, LIFR, OSMR, ZNF264 and ZNF543 genes in human plasma DNA based on digital PCR technology. Compared with the existing detection methods for detecting gene methylation based on the MethyLight technology, the detection method of the present invention has higher detection sensitivity. Based on the biomarkers screened in the present invention, an attempt can be made to establish a rapid, sensitive and specific detection method system for colorectal cancer diagnosis, which provides help for large-scale population screening and auxiliary colorectal cancer clinical diagnosis.

Description of drawings

Figure 1 shows the average methylation levels of 15 colorectal cancer-specific methylated CpG sites in 29 types of tumor tissues, including colorectal cancer, and colorectal cancer peripheral blood leukocyte DNA;

Note: The WBC at the bottom of Tumor Tissues is the average methylation level of peripheral blood leukocyte DNA in colorectal cancer patients, while the WBC at the bottom of Normal Tissues is the average methylation level of peripheral blood leukocyte DNA in healthy controls;

Figure 2 shows the amplified fragments and signal distributions included in three multiplex digital PCR detection systems (Multiplex ddPCR, mddPCR);

Among them: (A) mddPCRassay 1, (B) mddPCR assay 2 and (C) mddPCR assay 3.

Note: Each single amplification system or multiple amplification system amplifies four templates, including positive control material M, negative quality control material U, RNase-free water and non-template control NTC;

Figure 3 is a comparative analysis of the detection sensitivity of multiplex digital PCR system 1 and multiplex MethyLightPCR (mqPCR) system 1;

Among them: (A) the relationship between the Cq value of the amplification curve of the mqPCR assay 1 detection system and the log2 (standard methylation level); (B) the relative methylation level of the mddPCR assay 1 detection system (target gene copy number) (C) Droplet distribution of mddPCR assay 1 amplifying serial dilution standards, solid line is the manually set threshold for dividing positive and negative droplets ;

Figure 4 is a comparative analysis of the detection sensitivity of the multiplex digital PCR system 2 and the multiplex MethyLightPCR system 2;

Among them: (A) the relationship between the Cq value of the amplification curve of the mqPCR assay 2 detection system and the log2 (standard methylation level); (B) the relative methylation level of the mddPCR assay 2 detection system (target gene copy) (C) Droplet distribution of mddPCR assay 2 amplified dilution standard, the solid line is the manually set threshold for dividing positive and negative droplets ;

5 is a comparative analysis of the detection sensitivity of the multiplex digital PCR system 3 and the multiplex MethyLightPCR system 3;

Among them: (A) the relationship between the Cq value of the amplification curve of the mqPCR assay 3 detection system and the log2 (standard methylation level); (B) the relative methylation level of the mddPCR assay 3 detection system (target gene copy) (C) Droplet distribution of mddPCR assay 3 amplification serial dilution standard, the solid line is the manually set threshold for dividing positive and negative droplets ;

Figure 6 shows the diagnostic performance of three multiplex digital PCR detection systems.

Among them: (A) Box plots of the number of methylated molecule copies per milliliter of plasma detected by three multiplex digital PCR systems; (B-D) ROC curves of three multiplex digital PCR systems to differentiate colorectal cancer and healthy controls.

Detailed ways

The present invention will be described in detail below through specific embodiments, so that those skilled in the art can easily implement the present invention according to the disclosure of the present specification. The embodiments described below are only preferred examples of the present invention, and are not intended to limit the present invention in other forms. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1 Screening and verification of cfDNA methylation markers in colorectal cancer

1. Colorectal cancer tissue specificity

The invention utilizes databases such as TCGA to analyze the colorectal cancer tissue-specific methylation profile, screen out colorectal cancer-specific methylation markers, and screen out B3GALNT1, C6orf97, FAM72A, FAM72B, LIFR, OSMR, ZNF264 and 15 CpG sites on the ZNF543 gene. Based on the 450K methylation data of 29 cancer tissues including colorectal cancer in public databases such as TCGA and GEO, and the 450K methylation data from colorectal cancer and peripheral blood leukocytes of healthy people, it is shown that 15 CpG sites are in colorectal cancer tissues. Showing specific hypermethylation, the average methylation level is shown in Figure 1, and the genomic information of the 15 CpG sites is shown in Table 1. Using 395 colorectal cancer tissues and 45 paracancerous tissues in the TCGA database, and three datasets from GEO (GSE48684, GSE101764, and GSE131013), a total of 270 colorectal cancer tissues and 334 paracancerous tissues were used for evaluation. The diagnostic effect of 15 CpG sites, ROC curve analysis results show that the AUC range of 15 CpG sites is 0.643-0.903, and the sensitivity and specificity of diagnosing colorectal cancer are between 43.5%-80.3% and 91.0%-98.9%, respectively The detailed results are shown in Table 2.

Based on the above results, the 15 CpG sites screened by the present invention are colorectal cancer tissue-specific hypermethylation sites, and have certain diagnostic capabilities.

Table 1 Genome information of tissue-specific methylated CpG sites in colorectal cancer

Note: Refer to 450K chip annotation information for genome location (GRCh37/hg19)

Table 2. Diagnostic performance of 215 CpG loci in differentiating colorectal cancer from paracancerous tissue samples in four datasets

Embodiment 2 Multiple ddPCR detection method and establishment of test kit

The present invention adopts droplet digital PCR (droplet digital PCR, ddPCR) to quantitatively detect the methylation level of candidate markers in plasma cfDNA.

1. Primer and probe design

Following the conventional MethyLight primer and probe design principles, a total of 7 amplified fragments were successfully designed, including 10 candidate CpG sites located on FAM72A, FAM72B, LIFR, OSMR, ZNF264 and ZNF543 genes. The amplification primers include universal/methyl Sequence amplification primers and internal reference gene sequence amplification primers, TaqMan probes include methylation probes and internal reference gene probes, nucleotides for connecting reporter fluorescent groups and quenching groups, methylation probes and The internal reference gene probe should use different fluorescent groups such as FAM, VIC, etc. The specific reference to the fluorescence range of the digital PCR machine, the quenching group can choose the MGB group that can increase the annealing temperature of the probe, or use the padlock probe; ddPCR reaction The primer and probe sequences are shown in Table 3. In the sequence, Y represents the degenerate base T/C; R represents the degenerate base A/G.

Table 3 Primer and probe sequences of digital PCR detection system

In the exemplary embodiment of the present invention, the main feature of the ddPCR technology used is that the PCR mixture is added to the droplet generator, and each reaction well generates about 10,000 to 20,000 droplets, and each droplet contains 0- For 1 copy of the target gene fragment or background DNA, the droplet is determined to be positive or negative according to the end-point signal after PCR amplification, and the copy number in the original template is calculated based on the Poisson distribution. Unlike traditional methylation-specific PCR, it is not affected by PCR inhibitors and does not rely on standard curve quantification, enabling absolute quantification of rare methylation events in low-concentration DNA samples. In each PCR layout, the positive quality control substance (M), negative quality control substance (U), RNase-free water H ₂ O and no-template control NTC should be included at the same time. Bisulfate-treated fully methylated human genomic DNA, while the negative control can be sulfurized, unmethylated human genomic DNA. According to the threshold value set by the quality control material, H ₂ O and NTC, determine whether the sample to be tested contains the DNA target molecules of the methylated human FAM72A, FAM72B, LIFR, OSMR, ZNF264 and ZNF543 genes. Number of target DNA methylated molecules per ml of plasma for human FAM72A, FAM72B, LIFR, OSMR, ZNF264 and ZNF543. Data analysis is carried out by corresponding already commercial software (eg QuantaSoft, Bio-Rad).

2. DNA sample extraction and preparation

The DNA template detected by the present invention can be derived from the extraction of circulating cell-free DNA (cfDNA), circulating tumor DNA (ctDNA) in the peripheral blood plasma/serum of the subject or by extracting the DNA of circulating tumor cells; the specific operation is detailed as follows.

1) Extraction of cfDNA/ctDNA from the sample to be tested

A total of 368 blood samples were collected in the present invention, including 195 colorectal cancer patients, 22 adenoma patients, 5 polyp patients, 102 healthy controls, 20 breast disease patients (including 14 breast cancer patients) and 24 lung cancer patients. Disease patients (18 cases of lung cancer), the basic information is shown in Table 4. Obtain 3-5mL of plasma or serum from each sample, and use a commercial cell-free nucleic acid extraction kit such as QIAamp Circulating Nucleic Acid Kit to extract DNA samples from plasma or serum. The specific operation is performed according to the kit's operating manual.

Table 4 Clinical and demographic characteristics of blood samples

2) Sulfation of the DNA of the sample to be tested

The extracted DNA to be tested was treated with bisulfite using a commercial kit commonly used in the market, such as EZ DNAMethylation-Gold ^TM Kit, and the specific operation was carried out according to the kit operation manual.

3. Multiplex droplet digital PCR (mddPCR) detection system

Use QX200 ^TM Droplet Digital ^TM PCR System (Bio-Rad) detection system for mddPCR detection, the specific steps refer to the operation manual. In order to improve the utilization of cfDNA samples and the sensitivity of detection, multiple targeted amplification fragments were combined into the same reaction well to establish a multiplex digital PCR (Multiplex ddPCR, mddPCR) detection system, which was limited by the choice of the fluorescence channel of ddPCR (Dual channel, FAM and VIC), the FAM fluorophore is used to label all target site methylation probes, and the VIC fluorophore is used to label the internal reference gene probe (ACTB). According to the signal value distribution of positive droplets and negative droplets of methylation standards in different amplified fragment systems, three multiple amplification systems were successfully constructed by combining 7 amplified fragments (Fig. 2): mddPCR assay 1 contains ZNF543, ZNF264 and LIFR amplified fragments, mddPCR assay 2 includes amplified fragments FAM72A and FAM72B_2, and mddPCR assay 3 includes amplified fragments OSMR and FAM72B_1. During the experiment, the concentration ratio of primers and probes in the system may be changed according to the space temperature and operation time, and the corresponding droplet signal distribution and threshold line fluctuate within a reasonable range. In addition, the fluorescence background and amplification effect of probes synthesized in different batches (some specific sequence probes labeled with MGB) may be quite different. Gradient PCR determines optimal annealing temperature.

(1) mddPCR reaction system: Divide the sulfurized sample into three parts, and configure three multiplex digital PCR reaction systems respectively. The working concentrations of primers and probes are 20 μM and 10 μM, respectively. The probe solution should be protected from light, and the total reaction volume is 21 μL, the specific system preparation is shown in Table 5.

Three mddPCR detection reaction systems of table 5 are prepared

(2) Preparation and amplification of the droplet PCR reaction system: Add the prepared system to the 8 holes in the middle row of the droplet generating card, add 70 μL of droplet generating oil to the empty hole below, and put a rubber pad on it. Slowly transfer to a droplet generator to generate droplets, generating 10,000-20,000 droplets in about 2 minutes. Pipette 40 μL of the prepared droplets and slowly transfer them to a 96-well plate. After sealing the membrane, the PCR reaction should be performed within 30 minutes, or placed in a 4°C refrigerator for PCR within 4 hours. The PCR reaction was carried out in the instrument according to the set reaction conditions.

The above PCR amplification conditions were pre-denaturation at 95°C for 10 minutes, denaturation at 94°C for 30 seconds, annealing at 60°C for 60 seconds, a total of 40 cycles, inactivation of the enzyme at 98°C for 10 minutes, and then maintained at 4°C; °C/s.

(3) Sensitivity evaluation of multiplex digital PCR detection

In order to evaluate the detection sensitivity of the mddPCR system, the diluted M standard (2.5ng/μL) and U standard (2.5ng/μL) were prepared into 100%, 20%, 4%, 0.8%, 0.16% according to different volumes , 0.032%, 0.006% and 0% methylation level standard series, the total quality of DNA loading is controlled at about 10ng; the same standard series is also detected on multiplex MethyLight PCR (Multiplex MethyLight, mqPCR), all standard Both product series were tested repeatedly four times to compare the detection performance of the two. Except that the detection reagent is KAPA PROBE FAST qPCR Master Mix (2X) Kit (Roche), the rest of the system preparation is the same as that of digital PCR. Methylation detection was performed on the LightCycler 480II real-time fluorescence quantitative PCR instrument. After the experiment was completed, the Abs Quant/Fit Points module of Roche was used to analyze the experimental results. The experimental steps and experimental operating conditions refer to digital PCR. As shown in Figures 3-5, the multiplex digital PCR detection system constructed in the present invention can detect methylation levels as low as 0.032%, and its detection sensitivity is 5-125 times higher than that of multiplex MethyLight PCR.

(4) Signal detection and result judgment: After PCR amplification, put the 96-well plate into the microdroplet reader, draw the microdroplets of each sample in the set order, and pass the two-color detector with the microdroplet reading oil. (Bio-Rad), the number of copies of methylated molecules per milliliter of plasma in the sample to be tested was calculated by QuantaSoft (version 1.7) software analysis.

4. Judgment criteria for the effectiveness of the testing system and results

Judgment of validity and results of multiplex digital PCR detection system:

a) Positive quality control material (M): If there are 2 or more positive droplets in FAM (Ch1) channel and VIC (Ch2) channel, and no more than 1 positive droplet in RNase-free water/NTC, it is judged as Positive controls are valid. Otherwise reformulate.

b) Negative quality control substance (U): only if there are 2 or more positive droplets in the VIC (Ch2) channel, and no more than 1 positive droplet in the FAM (Ch1) channel, RNase-free water/NTC, it is judged that Negative controls are effective. Otherwise reformulate.

c) Judgment of results: The threshold line is delineated according to the positive and negative quality control substances, RNase-free water/NTC, the samples with the total number of droplets less than 8000 or no positive droplets in the VIC channel are rejected, and there are at least 2 samples in the FAM channel Positive droplets above the threshold line were considered as methylation positive samples, and the number of methylated molecule copies per milliliter of plasma (copies/mL) was calculated.

5. Test results and analysis

As shown in Figure 6, the copy number of methylated molecules detected in blood samples of colorectal cancer patients was significantly higher than that of healthy controls and non-colorectal cancer patients. The results of ROC curve analysis showed that the AUCs of mddPCR assay 1, mddPCR assay 2 and mddPCRassay 3 to distinguish colorectal cancer from healthy controls were 0.769 (95%CI: 0.730-0.809), 0.842 (95%CI: 0.807-0.878) and 0.774 ( 95% CI: 0.736-0.813). A total of 113 colorectal cancer patients were judged to be positive in mddPCR assay 1, and the sensitivity was 57.9%. The sensitivity of mddPCRassay 2 and mddPCR assay 3 were 71.8% and 57.9, respectively. %, the corresponding specificities were 93.1%, 93.1% and 96.1%, respectively. The sensitivity of the three detection systems increased with the increase of TNM stage, ranging from 35.9% to 94.4%. In addition, the three detection systems also have certain diagnostic capabilities in patients with early-stage colorectal cancer adenomas and polyps, and the sensitivity in adenomas ranges from 22.7% to 31.8% (Table 6).

Table 6 The methylation positive rate of three multiplex digital PCR detection systems in cfDNA of colorectal cancer patients

The number of copies of methylated molecules detected per milliliter of plasma in the three systems was summed to evaluate their combined diagnostic effect. The combined analysis results of the three systems were named as combined_7 (a total of 7 detection fragments), and the cutoff value was set to 0. A total of 165 patients with 195 colorectal cancer patients were judged to be positive, the sensitivity was 84.6%, and the corresponding specificity was 84.3% (Table 7); its false positive rates in breast and lung disease patients were 45% and 50%, respectively. Considering that mddPCR assay 1 has a high false positive rate in non-colorectal cancer patients, the combined results of mddPCR assay 2 and mddPCR assay 3 were named combined_4 (4 detection fragments in total), and the sensitivity and specificity of diagnosing colorectal cancer were 78.5% and 89.2%, respectively; the misdiagnosis rates in breast and lung diseases were 30.0% and 33.3%, respectively.

Table 7 Methylation positive rate of multi-system combined analysis in colorectal cancer cfDNA in the present invention

The preferred specific embodiments and implementation examples of the present invention have been described in detail above, but the present invention is not limited to the above-mentioned implementation examples and implementation examples. Within the scope of knowledge possessed by those skilled in the art, the invention can also be used without departing from the concept of the present invention. make various changes.

Claims (7)

1. Specific methylation markers related to colorectal cancer, wherein the methylation markers are B3GALNT1, C6orf97, FAM72A, FAM72B, LIFR, OSMR, ZNF264 or ZNF543 gene sequences containing at least one CpG methylation site or fragments thereof, or any combination of the gene sequences or fragments thereof;

genome annotation information according to the 450K chip (GRCh37/hg19), in which 1 CpG site, cg20841906, is present on the B3GALNT1 gene at the 160822911 th nucleotide of the 3 rd chromosome complement;

wherein 1 CpG site, cg00117463, is present on C6orf97 gene at nucleotide 151815241 of the positive strand of chromosome 6;

wherein 2 CpG sites are respectively presented on FAM72A gene, namely cg00552973 and cg10439914, wherein cg00552973 is located at the 206137054 th nucleotide of the 1 st chromosome reverse strand, and cg10439914 is located at the 206137759 th nucleotide of the 1 st chromosome positive strand;

wherein 6 CpG sites are present in FAM72B gene, cg07784526, cg18948743, cg08728856, cg00198436, cg07236150 and cg09169215, wherein cg07784526 is located at the 120838320 th nucleotide of the plus strand of chromosome 1, cg18948743 is located at the 120838323 th nucleotide of the plus strand of chromosome 1, cg08728856 is located at the 120838718 th nucleotide of the plus strand of chromosome 1, cg00198436 is located at the 120839380 th nucleotide of the minus strand of chromosome 1, cg07236150 is located at the 120839385 th nucleotide of the minus strand of chromosome 1, and cg09169215 is located at the 120839390 th nucleotide of the minus strand of chromosome 1;

wherein 1 CpG site, cg18174928, is located at 38557085 th nucleotide of the No. 5 chromosome plus strand on the LIFR gene;

wherein 1 CpG site, cg17528648, is present in the OSMR gene at nucleotide 38846100 of the plus strand of chromosome 5;

wherein 2 CpG sites are respectively cg26970847 and cg11140785 on the ZNF264 gene, wherein cg26970847 is positioned at 57702793 th nucleotide of the 19 th chromosome reverse strand, and cg11140785 is positioned at 57703301 th nucleotide of the 19 th chromosome reverse strand;

wherein 1 CpG site, namely cg14786398, is positioned at 57831816 th nucleotide of the reverse chain of No. 19 chromosome on ZNF543 gene.

2. A digital PCR kit for detecting methylation of genes related to colorectal cancer is characterized by comprising a combination of digital PCR primers and probes for detecting CpG methylation sites of genes related to colorectal cancer, such as FAM72A, FAM72B, LIFR, OSMR, ZNF264 and ZNF543, wherein the kit also comprises a primer pair and a probe required by an amplified fragment of an internal reference gene ACTB gene of which the sample loading amount needs to be corrected, and the sequences of the primers and the probes are as follows:

wherein, FAM72A primer pair is used for amplifying FAM72A gene fragment, and the probe is used for detecting 1 CpG site existing on the gene fragment, namely cg00552973, which is located at 206137054 th nucleotide of the 1 st chromosome reverse strand; the FAM72B _1 primer pair is used for amplifying a FAM72B gene fragment, the probe is used for detecting 2 CpG sites existing on the gene fragment, namely cg07784526 and cg18948743, the cg07784526 is positioned at the 120838320 th nucleotide of the 1 st chromosome positive strand, and the cg18948743 is positioned at the 120838323 th nucleotide of the 1 st chromosome positive strand; the FAM72B _2 primer pair is used for amplifying FAM72B gene fragments, probes are used for detecting 3 CpG sites existing on the gene fragments, namely cg00198436, cg07236150 and cg09169215, wherein cg00198436 is positioned at the 120839380 th nucleotide of the 1 st chromosome reverse strand, cg07236150 is positioned at the 120839385 th nucleotide of the 1 st chromosome reverse strand, and cg09169215 is positioned at the 120839390 th nucleotide of the 1 st chromosome reverse strand; the LIFR primer pair is used for amplifying an LIFR gene fragment, and the probe is used for detecting 1 CpG locus existing on the gene fragment, namely cg18174928, which is positioned at 38557085 th nucleotide of the plus strand of No. 5 chromosome; the OSMR primer pair is used for amplifying an OSMR gene fragment, and the probe is used for detecting 1 CpG locus existing on the gene fragment, namely cg17528648, and is positioned at 38846100 th nucleotide of the No. 5 chromosome positive strand; the ZNF264 primer pair is used for amplifying ZNF264 gene fragments, and the probe is used for detecting 1 CpG locus existing on the gene fragments, namely cg11140785, which is positioned at 57703301 th nucleotide of the 19 th chromosome reverse strand; the ZNF543 primer pair is used for amplifying ZNF543 gene segment, and the probe is used for detecting 1 CpG locus existing on the gene segment, namely cg14786398, which is located at 57831816 th nucleotide of No. 19 chromosome reverse strand.

3. The digital PCR kit of claim 2, wherein the 5 'end of the probe comprises a fluorescent reporter group, the fluorescent reporter group comprises any one of FAM, VIC/HEX, NED, ROX, TET, JOE, TAMRA, CY3 and CY5, the methylated probe and the reference gene probe for CpG methylation site detection use different fluorescent groups, the 3' end of the probe comprises a fluorescence quenching group, and the fluorescence quenching group comprises any one of MGB, BHQ-1, BHQ-2 and BHQ-3.

4. The digital PCR kit of claim 2, wherein the combination of 7 amplified fragments is three multiplex digital PCR detection systems, the multiplex ddPCR system 1 is used for amplifying LIFR, ZNF264 and ZNF543 fragments, the multiplex ddPCR system 2 is used for amplifying FAM72A and FAM72B _2 fragments, and the multiplex ddPCR system 3 is used for amplifying FAM72B _1 and OSMR fragments; the digital PCR reaction system and conditions of the kit are as follows:

digital PCR reaction system: 10 mu L of PCR premix solution 2 XddPCR Supermix for Probes, the final concentration range of primers is between 0.2 mu M and 0.9 mu M, the final concentration of Probes is between 0.1 mu M and 0.5 mu M, 0.002 to 70ng/uL of template DNA, and the RNA-free enzyme water is supplemented to 21 uL;

the digital PCR reaction conditions were as follows:

pre-denaturation at 95 ℃ for 10 min, denaturation at 94 ℃ for 30 sec, annealing at 60 ℃ for 60 sec for 40 cycles in total, enzyme inactivation at 98 ℃ for 10 min, and then retention at 4 ℃; the temperature rising and falling speed is less than or equal to 2.5 ℃/s.

5. The digital PCR kit of claim 4, wherein the template DNA is DNA from the blood of a subject.

6. The digital PCR kit of claim 5, wherein the template DNA is circulating free DNA (cfDNA), circulating tumor DNA (ctDNA) or Circulating Tumor Cell (CTCs) DNA in peripheral blood plasma or serum.

7. Use of the digital PCR kit of any one of claims 2-6 for the preparation of a diagnostic reagent for colorectal cancer.

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