CN112430657B - Methylation marker related to colorectal cancer and kit for detecting colorectal cancer - Google Patents

Abstract

The invention discloses a colorectal cancer-related methylation marker and a kit for detecting colorectal cancer. The methylation markers are 10 CpG sites distributed on ZNF304, LIFR and C20orf194 genes. The present invention detects the methylation levels of C20orf194, ZNF304 and LIFR genes in blood and tissue DNA for the first time, and uses the detection method combining qMSP and digital PCR and MethyLight for the first time. The reaction conditions have successfully established a detection method based on qMSP and ddPCR technology to detect the methylation of human ZNF304 and LIFR genes. Compared with the existing human SEPT 9 gene methylation detection method based on qPCR technology, the detection method of the present invention has higher specificity. The ddPCR method can qualitatively and quantitatively detect the methylation of ZNF304 and LIFR genes in colorectal cancer patients by calculating the number of methylation-positive droplets and the ratio of methylation-positive droplets to non-methylation-positive droplets. Auxiliary diagnosis of colorectal cancer.

Description

Colorectal cancer-associated methylation markers and assays for colorectal cancer detection kit

technical field

The invention relates to a colorectal cancer-related methylation marker, and also relates to a kit for detecting colorectal cancer, which belongs to the technical field of biomedicine.

Background technique

Colorectal cancer is a common malignant disease of the digestive tract. According to GLOBCAN 2018, it is estimated that there will be more than 1.8 million new cases, accounting for about 10% of new cancer cases each year, ranking third in the world, and the mortality rate is second only to lung cancer. ^[1] . It is estimated that there will be more than 140,000 new cases and 50,000 deaths in the United States in 2019, accounting for about one-tenth of all cancer incidence and deaths ^[2] . The 5-year survival rate of colorectal cancer can reach more than 65%, and the 5-year survival rate of patients with early diagnosis and treatment can reach more than 80%, while the 5-year survival rate of stage IV patients is only 12% ^[3] . According to the statistics of the National Cancer Center from 2000 to 2011, the incidence and mortality of colorectal cancer in China ranked fifth, but the incidence showed a significant upward trend, and the growth rate of men has been higher than that of women ^[4] . Contrary to the situation in China, the incidence and mortality of colorectal cancer in the United States have shown a significant downward trend since 1990, which is closely related to the changes in American lifestyles and dietary patterns, especially the formulation of early screening strategies ^[5] . It can early detect and remove colorectal lesions in the precancerous or early stage, thereby avoiding the occurrence and progression of some tumors ^[6] , and the rapid decline in the incidence in recent years is mainly due to the use of colonoscopy. Adults aged 50 and above in the United States The use of colonoscopy in 2000 increased from 21% in 2000 to 60% in 2015, an increase of nearly three-fold; while the incidence of adults under the age of 50 is still showing a 2% annual growth rate ^[7] . Therefore, early screening is an effective measure to control colorectal cancer.

The early screening of colorectal cancer mainly includes fecal occult blood test (FOBT) and colonoscopy. The fecal occult blood test has the advantages of simplicity and non-invasiveness, but its sensitivity is low, the false positive rate is high, and the patient's compliance is poor ^[8] . Colonoscopy, as the current mainstream method for screening, diagnosing and treating colorectal cancer, has significantly reduced morbidity and mortality in the United States and Europe, but its invasiveness and adverse reactions, time-consuming, and high cost may be to some extent Limit its popularity ^[9] . And can only provide a static check result ^[10] . Serum carcinoembryonic antigen (CEA) has good specificity as a non-invasive biomarker to identify occult CRC, but is limited by its low sensitivity (40% to 60%) ^[11] .

Circulating tumor DNA (ctDNA) is mainly composed of primary tumors, circulating tumor cells (CTCs), and DNA fragments that have been minutely or significantly transferred to the blood of cancer patients. Most of ctDNA is derived from apoptosis and necrosis of tumor cells. ^[12] . Mandelp first discovered cell-free DNA (cfDNA) in normal human peripheral blood in 1948 ^[13] . In 1977, Leon SA et al again found that the plasma cfDNA content of tumor patients was significantly higher than that of healthy people, and the cfDNA level of advanced tumor patients was higher ^[14] . Until 1994, Sorenson, GD found RAS mutation in the blood of pancreatic cancer patients for the first time, and it was consistent with the tissue ^[15] . Subsequent studies have shown that ctDNA carries tumor-specific genetic changes, including mutation ^[16] , copy number variation ^[17] , and found that ctDNA also carries cancer tissue-specific epigenetic changes, and tumor tissue-specific methylation patterns are related to ctDNA is highly correlated ^[18] . Therefore, the detection of biological characteristics of ctDNA can achieve minimally invasive and rapid cancer screening and real-time longitudinal monitoring of cancer.

DNA methylation is one of epigenetic modifications involved in differentiation and development, aging, tumorigenesis, etc. Genome-wide hypomethylation and CpG island hypermethylation in promoter regions are the most common biological phenomena in tumor tissues ^[19] . Aberrant methylation often results in defective gene expression, and hypermethylation of tumor suppressor genes is an early event in many tumors and may be one of the earliest detectable tumor alterations associated with tumorigenesis ^[20,21] . Compared with mutation detection, methylation has advantages in cancer screening and diagnosis, including higher clinical sensitivity and dynamic range, multiple detectable methylation target regions, and within each target genomic region Multiple altered CpG sites ^[22] . Multiple CpG loci in each selected region can be detected in combination to obtain "cancer-specific methylation patterns." In addition, it has been shown that methylation analysis can be used to distinguish tissue origin and cancer subtype ^[23] . Therefore, methylation has more advantages as a tumor diagnostic marker than gene mutation, and is one of the biomarkers with great potential for tumor-specific diagnosis.

Plasma SEPT9 promoter methylation has been extensively studied in colorectal cancer, and studies have shown that SEPT9 methylation has a sensitivity of 48.2% to 95.6% and a specificity of 69% to 97.1 for the diagnosis of colorectal cancer. % between ^[24] . It has been proven to be a highly sensitive and specific biomarker for colorectal cancer plasma detection and was approved by the US Food and Drug Administration (FDA) in 2016 ^[25,26] . However, SEPT9 is also hypermethylated to varying degrees in the plasma of other cancers such as lung cancer ^[27] , head and neck cancer ^[28] , and gastric cancer ^[29] . Therefore, ctDNA-specific methylation biomarkers for early diagnosis of colorectal cancer remain to be further investigated.

The proportion of ctDNA in cfDNA varies from 0.01% to 60%, and its concentration correlates with tumor volume, stage, vascularization, proliferation rate and cell death rate ^[30] . Therefore, it is a great challenge to achieve sensitive and reproducible detection of ctDNA methylation levels. The current SEPT9 detection kit uses a detection method that combines methylation-specific PCR (MSP) with TaqMan probes, called MethyLight, which can be used in a relative manner after correcting for DNA load and normalizing methylated DNA standards. Quantifying the amount of methylated alleles greatly improves the ability to detect and quantify methylated alleles ^[31] . However, MethyLight is still susceptible to PCR inhibitors, has limited sensitivity to detect rare methylated alleles in a background of unmethylated alleles, and can be biased during normalization, which can lead to inconsistent results, especially is in the detection of low levels of DNA methylation (including low concentrations and low molecular weight). Therefore, conventional methylation-specific quantitative PCR usually cannot achieve the sensitivity to accurately detect low-level DNA methylation as a cancer biomarker ^[32] . Digital PCR (droplet digital PCR) is a technique for absolute quantification of nucleic acid molecules, which was originally defined as single-molecule template PCR amplification, which separates a sample into individual molecules and amplifies them by PCR; negative and Positive, combined with Poisson distribution to calculate the copy number in the original sample ^[33] ; digital PCR is not affected by amplification efficiency and PCR inhibitors ^[34] . Studies have shown that digital PCR can detect 0.001% of target gene mutations from background DNA ^[35] . Ming et al. demonstrated that MethyLight ddPCR has a 25-fold lower limit of quantitation (LOQ) and a 20-fold lower limit of detection (LOD) compared to conventional MethyLight PCR ^[36] . Therefore, MethyLight ddPCR is a more suitable technique for detecting the methylation level of ctDNA.

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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 methylation marker 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 invention utilizes databases such as TCGA to analyze the tissue-specific methylation profile of colorectal cancer, and screens out tumor methylation-specific markers, mainly screening out 10 CpG sites distributed on ZNF304, LIFR and C20orf194 genes. Then, the targeted sequencing MethylTarget technology was used to verify the differential methylation in colorectal cancer and adjacent normal tissues, and based on the tissue results, a DNA methylation-specific quantitative detection technology (QuantitativeMethylation Specific PCR, qMSP) and digital PCR-based methods for detecting ZNF304 and LIFR gene methylation in human tissue/plasma DNA and the probes and primers used, compared with the existing human SEPT 9 gene methylation detection methods based on qPCR technology, The detection method of the present invention has higher specificity. In addition, digital PCR can perform absolute quantification of the target fragment to be detected, and obtain the methylation level of the target fragment in the sample to be tested by calculating the ratio of methylation and non-methylation positive droplets or copy numbers, which can achieve high-sensitivity analysis. detection.

Therefore, the present invention first proposes a methylation marker related to colorectal cancer, the methylation marker is a ZNF304, LIFR or C20orf194 gene sequence or a fragment thereof containing at least one CpG methylation site , or any combination of described gene sequences or fragments thereof;

According to the genome annotation information of the 450K chip, there are 3 CpG sites on the C20orf194 gene, cg04125300, cg15863924 and cg02893482, cg04125300 is located at the 3388262 nucleotide of the anti-strand of chromosome 20, and cg15863924 is located on chromosome 20. At nucleotide 3388269 of the anti-strand, cg02893482 is located at nucleotide 3388900 of the positive strand of chromosome 20;

Among them, there are three CpG sites on the LIFR gene, cg18174928, cg11841722 and cg12602374, cg18174928 is located at nucleotide 38557085 of the positive strand of chromosome 5, and cg11841722 is located at nucleotide 38557253 of the anti-strand of chromosome 5 , cg12602374 is located at nucleotide 38557162 of the positive strand of chromosome 5;

Among them, there are 4 CpG sites in ZNF304 gene, namely cg10157975, cg13788592, cg21627760 and cg23250910, cg10157975 is located at the 57862442 nucleotide of the positive strand of chromosome 19, and cg13788592 is located at the 57862612 nucleus of the anti-strand of chromosome 19. At nucleotides, cg21627760 is located at nucleotide 57862638 of the positive strand of chromosome 19, and cg23250910 is located at nucleotide 57862410 of the anti-strand of chromosome 19.

Further, the present invention also provides a primer set for amplifying the methylation marker of colorectal cancer, the primer set is respectively used to amplify ZNF304, LIFR and LIFR containing the CpG site. The primer pair composition of the C20orf194 gene fragment is as follows:

Among them, the ZNF304_1 primer pair is used to amplify the two CpG sites present on the ZNF304 gene, cg13788592 and cg21627760 respectively, cg13788592 is located at the 57862612 nucleotide of the reverse strand of chromosome 19, and cg21627760 is located at the positive strand of chromosome 19 The ZNF304_2 primer pair is used to amplify the other two CpG sites present on the ZNF304 gene, cg10157975 and cg23250910, respectively, cg10157975 is located at the 57862442 nucleotide of the positive strand of chromosome 19, cg23250910 Located at nucleotide 57862410 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 ddPCR for colorectal cancer-related gene ZNF304 and LIFR gene methylation detection Primer-probe combination, described ddPCR primer-probe combination is shown in 1) and/or 2) below:

1) ddPCR primers and probes for ZNF304 methylation detection and ddPCR primers and probes for ZNF304 non-methylation detection, described primers and probe sequences are as follows:

2) ddPCR primers and probes for LIFR methylation detection and PCR primers and probes for LIFR non-methylation detection, described primers and probe sequences are as follows:

Wherein, preferably, the 5' end of the probe contains a fluorescent reporter group, and the fluorescent reporter group includes any one of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY3, CY5, And methylated probe and non-methylated probe use different fluorescent groups, the 3' end of the fluorescent probe contains a fluorescence quenching group, and the fluorescence quenching group includes MGB, BHQ-1 , any one of BHQ-2 and BHQ-3.

Wherein, preferably, the digital PCR reaction system and conditions of the kit are as follows:

Digital PCR reaction system: 2×ddPCR Supermix for Probes 10uL, add primers and probes to make the final concentrations of primers and probes respectively reach the final concentration of 0.9uM and the final concentration of probes of 0.25uM, 5-6uL of 0.002-70ng/uL Template DNA, make up ddH2O 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, a total of 40 cycles, inactivating the enzyme at 98°C for 10 minutes, and then holding 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 (CTCs) DNA in peripheral blood plasma or serum.

Further, the present invention also provides a qMSP kit for colorectal cancer-related gene methylation detection, the kit includes qMSP primers for colorectal cancer-related gene ZNF304 and LIFR gene methylation detection Probe combinations, the qMSP primer probe combinations are shown in the following 1) and/or 2):

1) qMSP primers and probes for ZNF304 methylation detection, described primers and probe sequences are as follows:

ZNF304 methylation forward primer 5’-CGTTTTTTGTTGTTTTGGGTT-3’

ZNF304 methylation reverse primer 5'-AAATACACGAACCGAACATAAA-3'

ZNF304 methylation probe 5'-AATACGTCACGACGACGACGACCA-3'

2) qMSP primers and probes for LIFR methylation detection, described primers and probe sequences are as follows:

LIFR methylation forward primer 5’-GGATCGCG TTTTTTCGAATG-3’

LIFR methylation reverse primer 5'-ACAAAAACGACGAAAACACG-3'

LIFR methylation probe 5’-TCCCCGCTTTCCTCTCCTCAAAACAC-3’

Wherein, preferably, the 5' end of the probe contains a fluorescent reporter group, and the fluorescent reporter group includes any one of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY3, CY5, The 3' end of the fluorescent probe contains a fluorescence quenching group, and the fluorescence quenching group includes any one of MGB, BHQ-1, BHQ-2, and BHQ-3.

Wherein, preferably, the PCR reaction system and conditions of the kit are as follows:

PCR amplification conditions: pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, a total of 45 cycles, cooling at 40°C for 30 seconds, and then maintained at 4°C.

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

1. The present invention discovered 10 colorectal cancer-specific CpG sites distributed on C20orf194, ZNF304, and LIFR genes for the first time by analyzing the methylation 450K high-throughput chip data. Eight of them were verified, and a detection method based on qMSP technology to detect the methylation of ZNF304 and LIFR genes in human tissue was successfully established.

2. The present invention detects the methylation levels of C20orf194, ZNF304 and LIFR genes in peripheral blood DNA for the first time, and uses the detection method combining digital PCR and MethyLight for the first time. According to the reaction conditions, a detection method based on ddPCR technology to detect the methylation of human ZNF304 and LIFR genes was successfully established. Compared with the existing human SEPT9 gene methylation detection method based on qPCR technology, the detection method of the present invention has higher specificity. The ratio of the methylation-positive droplets can then achieve absolute quantification, which can specifically qualitatively and quantitatively detect the methylation of ZNF304 and LIFR genes in patients with colorectal cancer, thereby realizing the auxiliary diagnosis of colorectal cancer.

Description of drawings

Figure 1 shows the average methylation levels of 11 cancer tissues from the TCGA database and the data of healthy human peripheral blood leukocytes from GEO;

Figure 2A and Figure 2B are box plots of site methylation levels contained in the C20orf194 gene;

Figure 3A and Figure 3B are box plots of site methylation levels contained in the LIFR gene;

Figure 4A and Figure 4B are box plots of site methylation levels contained in the ZNF304 gene;

Figure 5 shows the results of differential methylation analysis by targeted sequencing of CRC cancer and adjacent tissues;

Figure 6 shows the results of differential methylation analysis of CRC cancer, adenoma, polyp tissue and paracancerous tissue;

Fig. 7A and Fig. 7B are respectively the primer position map of amplifying ZNF304 gene and gene LIFR;

Wherein, the position of each CPG in FIG. 7A and the corresponding number are as follows:

Wherein, the position of each CPG in FIG. 7B and the corresponding number are as follows:

Figure 8 is the ZNF304 qMSP amplification standard curve;

Fig. 9 is LIFR qMSP amplification standard curve;

Figure 10 is the standard curve of ACTB qMSP amplification;

Figure 11 is a box plot of qMSP amplification of ZNF304 methylation levels;

Figure 12 is a box plot of qMSP amplified LIFR methylation levels;

Figure 13 is the test result of ZNF304 digital PCR quality control product;

Figure 14 shows the detection results of LIFR digital PCR quality control products;

Figure 15 is a box plot of the methylation levels of ZNF304 in the plasma of colorectal cancer patients and healthy controls;

Figure 16 is a box plot of LIFR methylation levels in the plasma of colorectal cancer patients 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 some, but not all, embodiments of the present invention. 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 present invention utilizes databases such as TCGA to analyze colorectal cancer tissue-specific methylation profiles, screen out tumor methylation-specific markers, and screen out 10 CpG sites mainly distributed on ZNF304, LIFR and C20orf194 genes. Based on the 450K methylation data of 11 cancer tissues including colorectal cancer in the TCGA database and healthy human peripheral blood leukocytes from GEO, the 10 CpGs were shown to be tissue-specific for colorectal cancer. The average methylation levels are shown in Figure 1. Box plots of site methylation levels included in the three genes are shown in Figure 2, Figure 3, and Figure 4. The genome information of the 10 CpG sites is shown in Table 1, and the cancer types are shown in Table 2.

Table 1

Note: The genome annotation information is taken from the 450K chip (GRCh37/hg19)

Table 2

2. Methylation-targeted sequencing

The tissue methylation detection method is a combination of target region enrichment and next-generation sequencing technology MethylTarget, which realizes multiplex PCR with high sequencing depth and can accurately calculate the methylation level of each CpG site. It is convenient to evaluate the methylation level and differential methylation level of the 10 CpG sites selected in the present invention. The technology is highly accurate, flexible, and cost-effective.

DNA Mini Kit(Qiagen，51306，德国)提取组织中的DNA，具体操作按照试剂盒操作手册进行。用Thermo ScientificNanoDrop 2000c分光光度计对提取的DNA进行定性和定量检测(A260/A280在1.7-1.9之间，A260/A230>2)。提取后DNA分装于-80℃保存。A total of 276 patients with primary colorectal cancer confirmed by histopathology were included in the present invention, including 8 adenoma tissues, 10 polyp tissues, and 23 partially matched adjacent tissues; the phenol-chloroform method and

DNA Mini Kit (Qiagen, 51306, Germany) was used to extract DNA from tissues, and the specific operations were carried out according to the kit operation manual. The extracted DNA was qualitatively and quantitatively detected with a Thermo Scientific NanoDrop 2000c spectrophotometer (A260/A280 between 1.7-1.9, A260/A230>2). After extraction, the DNA was aliquoted and stored at -80°C.

Fast DNA Bisulfite Kit(Qiagen，59826，德国)对1-2ug原始DNA进行亚硫酸氢盐处理，具体操作按照试剂盒操作手册进行。硫化后DNA用NanoDrop 2000c进行浓度(ng/ul)和纯度测定，硫化后DNA序列中非甲基化的C被转化为U，因此大多数序列不再互补而成为单链，与RNA较为相似。因此测定模式为RNA，检测结果A260/280在2.0-2.4之间且A260/A230>1提示硫化后DNA质量合格。硫化后DNA稀释至20-25ng/uL分装后于-20℃保存。use

The Fast DNA Bisulfite Kit (Qiagen, 59826, Germany) was subjected to bisulfite treatment on 1-2ug of the original DNA, and the specific operation was carried out according to the operation manual of the kit. The concentration (ng/ul) and purity of DNA after sulfidation were determined with NanoDrop 2000c. The unmethylated C in the sulfidized DNA sequence was converted to U, so most sequences were no longer complementary and became single-stranded, which was similar to RNA. Therefore, the assay mode is RNA, the detection result A260/280 is between 2.0-2.4 and A260/A230>1 indicates that the quality of DNA after sulfidation is qualified. After sulfuration, the DNA was diluted to 20-25ng/uL and stored at -20℃.

Methylation-targeted sequencing experimental steps:

1) Sample quality control

The concentration of DNA after sulfuration is between 20-25ng/uL, the total amount is ≥500ng, OD260/280=2.0-2.4, OD260/230≥1.

2) Primer design and optimization of single-site PCR conditions

Use primer3 or self-adjust to design high-quality sequencing primers for the region where the target CpG site is located. Before importing the sequence into the software, the target sequence must be manually converted to the post-sulfuration sequence, that is, the cytosine of the target CpG is kept unchanged and the C of the remaining CpG is converted to U; the human genome after bisulfite treatment can be selected as a template. , and the primers that amplified a clear single band were used for subsequent experiments. The C20orf194 gene contains a total of 3 CpG sites, and one site (3388900) was successfully pre-tested by primers; the three CpG sites contained in LIFR were pre-tested successfully; ZNF304 contained four CpG sites, which were designed on two primers respectively. The primer sequences are shown in Table 3.

table 3

3) Multiplex PCR primer panel optimization

The primers optimized in step 2) are mixed into a multiplex PCR primer panel, and the standard human genome is used as a template for amplification. Based on the results of capillary electrophoresis, determine whether each pair of primers in the multiplex system can efficiently and specifically amplify, and adjust accordingly to optimize the composition and concentration of primers in the multiplex PCR panel.

4) Multiplex PCR reaction of sample target fragment

The optimized multiplex PCR primer panel was used to carry out multiplex PCR amplification with the sulfurized DNA as the template. After quality control, mix the amplification products of all multiplex PCR primer panels using the same sample genomic DNA as the template, and ensure that the amount of each site primer amplification product is the same. 2 μL, diluted to 60 μL, as a template for the subsequent Index PCR step. The multiplex PCR reaction system is shown in Table 4 and the amplification conditions are shown in Table 5. The total reaction system is 10uL, and the rest is filled with water.

Table 4 Multiple reaction systems

component volume 10×buffer(TAKARA) 1μL dNTP (2.5mM) 1.2μL MgCl₂(25mM) 0.6μL Gene-specific primers (1 μM) 1μL HotTaq 5U/μL (Takara) 0.07μL Bisulfite-treated sample DNA 1μL ddH₂O 5.13μL total capacity 10μL

Table 5 Multiple reaction conditions

transsexual annealing extend Keep number of cycles 1Step 95℃2min 1× 2Step 95℃20s 62℃ 40s 72℃1min 11×(-0.5℃/cycle) 3Step 95℃20s 64℃30s 72℃1min 24× 4Step 72℃1min 1× 5Step 4℃ forever

5) Add a specific tag sequence to the sample

Using primers with Index sequences, specific tag sequences compatible with the illumina platform were introduced into the end of the library established after the amplification in the above steps by PCR amplification. The reaction used a PCR program of 12 cycles to minimize PCR bias. The IndexPCR system and amplification conditions are shown in Table 6 and Table 7. The total reaction system is 10uL, and the rest is made up with water.

Table 6

Table 7

transsexual annealing extend Keep number of cycles 1Step 95℃2min 1× 2Step 95℃20s 60℃ 40s 72℃1min 12 3Step 72℃2min 1× 4Step 4℃ forever

6) On-board sequencing after quantification

The Index PCR amplification products of all samples were mixed in equal amounts, and recovered by gel tapping to obtain the final MethylTarget sequencing library. The fragment length distribution of the library was verified by the Agilent 2100 Bioanalyzer. After the library molar concentration was accurately quantified, the Illumina Hiseq platform was finally used for high-throughput sequencing in a 2×150bp paired-end sequencing mode to obtain FastQ data.

3. Methylation-targeted sequencing data analysis

Firstly, the annotation information of the region amplified by the primers on the reference genome is checked according to the genomic location of the target region. Secondly, the sequencing raw data is sorted and the quality is evaluated, and the cytosine conversion efficiency is counted to evaluate the sulfur conversion efficiency. Finally, the methylation level of each CpG site was calculated.

C20orf194, ZNF304_1, ZNF304_2 are all designed on the reverse strand, so the position of the gene locus is increased by one bit compared to the 450K annotation information. Three samples with cytosine conversion efficiency <98% were excluded to ensure complete sulfuration conversion, 42 samples with sequencing depth less than 10× were excluded to ensure data quality, and the remaining 272 samples were data. The results of differential methylation analysis between CRC cancer and adjacent cancer are shown in Figure 5. The selected sites and their surrounding adjacent sites all show that the methylation level of cancer tissue is significantly higher than that of adjacent cancer tissue, and the AUC range is 46.17%-89.81 %, the results are shown in Table 8 (target sites are in bold); and the data results indicate that the screened genes may have an increasing trend in the methylation level at the stage of adenomatous polyps (see Figure 6 or Table 9). Based on external data and tissue verification results, it shows that the 10 CpG methylation sites screened in the present invention can be used as methylation sites for the specific detection of colorectal cancer, and the nucleosides containing the above CpG methylation sites Acid sequences can be used as methylation markers for the specific detection of colorectal cancer.

Table 8 MethylTarget_AUC

Table 9 MethylTarget_methylation

Example 2 Methylation-specific fluorescence quantitative PCR (qMSP)

The qMSP probe method was used for methylation detection of colorectal cancer, adjacent tissue and peripheral blood leukocyte DNA, and primers were designed based on the results of targeted sequencing. The target fragment plasmid was constructed with the fully methylated human genomic DNA treated with bisulfite as the template, and the methylation standard curve was constructed by gradient dilution and the equation was established. The ratio of the amplified copy number to that of the reference gene was used as the relative methylation level of the target gene.

1. Primer and probe design

Design qMSP primers and probes for ZNF304, LIFR and internal reference gene ACTB for the screened sites, amplification primers include methylation amplification primers and internal reference amplification primers, qMSP probes include methylation probes and internal reference probes; The internal reference primer was designed without CG sequence on the ACTB gene to prevent interference with methylation detection. The fluorescent groups of the probe, such as FAM, VIC, etc., refer to the fluorescence range of the fluorescence quantitative PCR instrument. The quenching group can be selected as the MGB group or others that can increase the annealing temperature of the probe; The primer-probe combination that obtained a clear, single and smooth "S"-shaped amplification curve was amplified in a system using hydrogen-salt-treated fully methylated human genomic DNA as a template for subsequent experiments. There was no fluorescent signal in NTC. The reaction primers and probe sequences are shown in Table 10. The primer positions are shown in Figure 7.

Table 10 Primer and probe sequences

2. Extraction and Preparation of DNA Samples

DNA Mini Kit(Qiagen，#51306，德国)提取样本中的DNA，具体操作按照试剂盒操作手册进行。用Thermo Scientific NanoDrop 2000c分光光度计对提取的DNA进行定性和定量检测(A260/A280在1.7-1.9之间，A260/A230>2)。提取后DNA分装于-80℃保存。A total of 90 cases of primary colorectal cancer (CRC) patients confirmed by histopathology, 41 cases of partially matched adjacent tissue (Normal), 52 cases of peripheral blood white blood cells (CRC_WB) of colorectal cancer patients, and peripheral blood of healthy controls were included in the present invention. White blood cells (WB) 50 cases; use

DNA Mini Kit (Qiagen, #51306, Germany) was used to extract DNA from samples, and the specific operations were carried out according to the kit's operating manual. The extracted DNA was qualitatively and quantitatively detected with a Thermo Scientific NanoDrop 2000c spectrophotometer (A260/A280 between 1.7-1.9, A260/A230>2). After extraction, the DNA was aliquoted and stored at -80°C.

Fast DNA Bisulfite Kit(Qiagen，#59826，德国)对1-2ug原始DNA进行亚硫酸氢盐处理，具体操作按照试剂盒操作手册进行。硫化后DNA用NanoDrop2000c进行浓度(ng/ul)和纯度测定，硫化后DNA序列中非甲基化的C被转化为U，因此大多数序列不再互补而成为单链，与RNA较为相似。因此测定模式为RNA，检测结果A260/280在2.0-2.4之间且A260/A230>1提示硫化后DNA质量合格。硫化后DNA稀释至20-25ng/uL分装后于-20℃保存。use

The Fast DNA Bisulfite Kit (Qiagen, #59826, Germany) bisulfite 1-2ug of the original DNA, and the specific operation was carried out according to the kit's operating manual. The concentration (ng/ul) and purity of DNA after sulfidation were determined with NanoDrop2000c. The unmethylated C in the sulfidized DNA sequence was converted to U, so most sequences were no longer complementary and became single-stranded, which was similar to RNA. Therefore, the assay mode is RNA, the detection result A260/280 is between 2.0-2.4 and A260/A230>1 indicates that the quality of DNA after sulfidation is qualified. After sulfuration, the DNA was diluted to 20-25ng/uL and stored at -20℃.

3. Human ZNF304, LIFR methylation fluorescent quantitative PCR detection system

480Probes Master PCR预混液及目的基因引物探针组合和模板见表11。使用

480Software release 1.5.0设定PCR反应扩增条件：95℃预变性10分钟，95℃变性10秒，60℃退火30秒，共进行45个循环，40℃冷却30秒，之后4℃保持。PCR完成后使用Abs Quant/2nd Derivative Max模块进行分析，将样本Cp值带入目标基因和内参基因标准曲线，计算相对甲基化水平。The reaction system includes

The 480Probes Master PCR master mix and target gene primer probe combinations and templates are shown in Table 11. use

480Software release 1.5.0 sets PCR amplification conditions: 95°C pre-denaturation for 10 minutes, 95°C denaturation for 10 seconds, 60°C annealing for 30 seconds, a total of 45 cycles, 40°C cooling for 30 seconds, and then 4°C holding. After the PCR is completed, the Abs Quant/2nd Derivative Max module is used for analysis, and the Cp value of the sample is brought into the standard curve of the target gene and the internal reference gene to calculate the relative methylation level.

Table 11

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

(1) Judgment of the effectiveness and results of the ZNF304 testing system:

a) Judgment of validity: the gradient dilution plasmid concentration range is 5E7-5E1 copies/ul, there are amplification signals and the amplification curve is smooth "S" shape, RNase-free water/NTC has no amplification signal or no "S" "type, the judgment is valid. Otherwise reconfigure.

b) Result judgment: The standard curve was constructed using the detection Cp value of the standard gradient dilution plasmid and the log-transformed concentration, and the amplification efficiency of the standard curve was between 1.9-2.1 (90%-110%). If the Cp value of the sample is within the range of the standard curve, it can be included in the standard curve to calculate the copy number; if the Cp value of the sample is greater than the Cp value of the lowest concentration plasmid, it indicates that the methylation of the human ZNF304 gene is negative or the degree of methylation is lower than the minimum detection limit. The ZNF304 amplification standard curve is shown in Figure 8.

(2) Judgment of the effectiveness and results of the LIFR testing system:

a) Judgment of validity: the gradient dilution plasmid concentration range is 5E7-5E1 copies/ul, there are amplification signals and the amplification curve is smooth "S" shape, RNase-free water/NTC has no amplification signal or no "S" "type, the judgment is valid. Otherwise reconfigure.

b) Result judgment: The standard curve was constructed using the detection Cp value of the standard gradient dilution plasmid and the log-transformed concentration, and the amplification efficiency of the standard curve was between 1.9-2.1 (90%-110%). The sample Cp value within the range of the standard curve can be included in the standard curve to calculate the copy number; the sample Cp value is greater than the lowest concentration plasmid Cp value, indicating that the human LIFR gene methylation is negative or the methylation degree is lower than the minimum detection limit. The LIFR amplification standard curve is shown in Figure 9.

(3) Judgment of the validity and results of the ACTB testing system:

a) Validity determination: The concentration range of the gradient dilution plasmid is 5E7-5E1 copies/ul, of which 5 consecutive concentrations have amplification signals and the amplification curve is a smooth "S" shape, and there is no amplification signal in RNase-free water/NTC If there is no "S" type, the judgment is valid. Otherwise reconfigure.

b) Result judgment: The standard curve was constructed using the detection Cp value of the standard gradient dilution plasmid and the log-transformed concentration, and the amplification efficiency of the standard curve was between 1.9-2.1 (90%-110%). If the Cp value of the sample is within the range of the standard curve, it can be included in the standard curve to calculate the copy number; if the Cp value of the sample is greater than the Cp value of the lowest concentration plasmid, it indicates that the human ACTB gene is negative or lower than the minimum detection limit. The ACTB amplification standard curve is shown in Figure 10.

5. Test results and analysis

The invention detects a total of 90 cases of primary colorectal cancer, 41 cases of partially matched adjacent tissues, 52 cases of peripheral blood leukocytes of colorectal cancer patients, and 50 cases of peripheral blood leukocytes of healthy controls; the sample Cp value is brought into the standard curve to calculate the relative methylation level. The results showed that ZNF304 and LIFR were hypermethylated in colorectal cancer tissues, but showed lower methylation levels in paracancerous and peripheral blood leukocytes, indicating that peripheral blood leukocyte DNA methylation levels did not affect the subsequent detection of the two genes in cfDNA. Interference, ZNF304 and LIFR box scatter plots are shown in Figure 11 and Figure 12 respectively. The sensitivity of ZNF304 methylation level in detecting colorectal cancer tissue was 77.8%, the specificity was 97.6%, and the area under the subject curve was 0.812; the sensitivity of LIFR methylation level in detecting colorectal cancer tissue was 73.3%, the specificity was 0.812 90.2% and the subject area under the curve was 0.806. The ROC curve was used to calculate the best cut-off value of the two genes, and the two genes and their combined positive rates were calculated in different stages. The results are shown in Table 12, Table 13, and Table 14.

Table 12

Table 13

sample LIFR positive/tested number Positive rate (%) Phase I 5/5 100% Phase II 27/38 71.1% Phase III 29/41 70.7% Stage IV 5/6 83.3% all patients 66/90 73.3% healthy control 4/41 9.8% (specificity 90.2%)

Table 14

sample ZNF304 and LIFR positive/tested numbers Positive rate (%) Phase I 5/5 100% Phase II 34/38 89.5% Phase III 36/41 87.8% Stage IV 6/6 100% all patients 81/90 90.0% healthy control 4/41 9.8% (specificity 90.2%)

Example 3 MethyLight ddPCR detection method and establishment of the kit

The technology used in the present invention is digital PCR technology combined with the MethyLight method. By designing specific primers and probes of the candidate methylation regions of human ZNF304 and LIFR, amplifying the bisulfite modified and prepared by microdroplet DNA samples were tested, and the ratio of the number of positive droplets amplified by PCR to the total number of positive droplets was used to determine whether the methylation and methylation levels of the candidate methylation regions of human ZNF304 and LIFR in the sample to be tested had occurred. Determine whether the sample to be tested has colorectal cancer.

1. Primer and probe design

Design ddPCR primers and probes for ZNF304 and LIFR for the screened sites, amplification primers include methylated sequence amplification primers and non-methylated sequence amplification primers, TaqMan probes include methylated probes and non-methylated sequence amplification primers The methylated probe, in order to connect the nucleotides of the reporter fluorescent group and the quenching group, the methylated probe and the unmethylated probe should use different fluorescent groups such as FAM, VIC, etc. Refer to the digital PCR machine for specific fluorescence The quenching group can choose the MGB group that can increase the annealing temperature of the probe, or use a padlock probe; the ddPCR reaction primers and probe sequences are shown in Table 15.

Table 15

In an exemplary embodiment of the present invention, the digital PCR employs droplet digital PCR (droplet digital, PCR). The main feature is that the digital PCR mixture is added to the droplet generator to generate 10,000 to 20,000 micro-reaction droplets for PCR amplification. In each PCR layout, the positive quality control substance (M), negative quality control substance (U), RNase-free water and blank control NTC should be included at the same time. The so-called positive quality control substance can be treated with bisulfite. fully methylated human genomic DNA, while the negative control can be sulfurized unmethylated human genomic DNA. According to the quality control material and blank control, the threshold is set to determine whether the sample to be tested contains the DNA target molecules of the methylated human ZNF304 and LIFR genes, and at the same time, the quantity of the methylated human ZNF304 and LIFR target DNA molecules is determined. Data analysis is performed by corresponding software already commercially available (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

Take 3-5 mL of plasma or serum from 40 patients with colorectal cancer diagnosed by histopathology or 13 healthy controls, and prepare DNA samples using a commercial free nucleic acid extraction kit such as QIAamp Circulating Nucleic Acid Kit. The specific operation is in accordance with the kit operation manual. Use 50uL of eluent to elute the DNA to be tested from the spin column, and use the Qubit3.0 fluorescence quantitative analyzer to measure the sample concentration. This system uses a specially developed fluorescent dye, which is more sensitive than the traditional UV absorption method, especially suitable for precious trace samples in the present invention.

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

Fast DNA Bisulfite Kit对提取的待测DNA进行亚硫酸氢盐处理，具体操作按照试剂盒操作手册进行。用20ul洗脱液洗脱待测DNA，用于数字PCR检测。Use commercially available kits such as

The Fast DNA Bisulfite Kit performs bisulfite treatment on the extracted DNA to be tested. The specific operation is carried out according to the kit operation manual. The DNA to be tested was eluted with 20ul of eluent for digital PCR detection.

3. Human ZNF304, LIFR gene methylation digital PCR detection system

(1) Digital PCR reaction system: Divide the sulfurized sample into two parts, and configure the reaction systems of the two genes respectively. In a 20uL PCR reaction system, 2×ddPCR Supermix for Probes 10uL, add primers and probes to make their final concentrations respectively To achieve a final primer concentration of 0.9uM and a final probe concentration of 0.25uM, 5-6uL of template DNA with a concentration of 0.002-70ng/uL, make up ddH2O to 21uL.

(2) Preparation and amplification of the droplet PCR reaction system: extract 20ul from the configured system (multiple systems were prepared in the previous step to avoid air bubbles) and add it to the 8 holes in the middle row of the droplet generating card (Bio-Rad). , add 70uL of droplet generating oil to the empty hole below, put a rubber pad on it, and slowly transfer it to a droplet generator (Bio-Rad) to generate droplets, and generate 10,000-20,000 droplets in about 2 minutes. Pipette 40uL 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 refrigerator at 4°C 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) Signal detection and result judgment: After PCR amplification, put the 96-well plate into the microdroplet reader, draw the microdroplets of each sample according to the set order, and pass the two-color detector with the microdroplet reading oil. (Bio-Rad), the number of droplets of ZNF304 and LIFR in the sample to be tested was calculated and analyzed by QuantaSoft (version 1.7) software.

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

(1) Judgment of the effectiveness and results of the ZNF304 testing system:

a) Positive quality control material (M): if there is a signal in the FAM (Ch1) channel, but no signal in the VIC (Ch2) channel and RNase-free water/NTC, the positive quality control material is judged to be valid. Otherwise reconfigure.

b) Negative quality control substance (U): There is no signal in the FAM (Ch1) channel, RNase-free water/NTC, and there is a signal in the VIC (Ch2) channel, and the negative quality control substance is judged to be effective. Otherwise reconfigure.

c) Determination of results: The threshold line is defined according to the positive and negative quality control substances, RNase-free water/NTC, and the number of positive droplets in the FAM (Ch1) channel is 2 or more positive for methylation; the VIC (Ch2) channel is positive Three or more droplets were judged as non-methylation positive. In this detection system, there are far more non-methylation positive droplets than methylation positive droplets. The test results of the quality control products are shown in Figure 13.

(2) Judgment of the effectiveness and results of the LIFR testing system:

a) Positive quality control material (M): if there is a signal in the FAM (Ch1) channel, but no signal in the VIC (Ch2) channel and RNase-free water/NTC, the positive quality control material is judged to be valid. Otherwise reconfigure.

b) Negative quality control substance (U): There is no signal in the FAM (Ch1) channel, RNase-free water/NTC, and there is a signal in the VIC (Ch2) channel, and the negative quality control substance is judged to be effective. Otherwise reconfigure.

c) Determination of results: The threshold line is defined according to the positive and negative quality control substances, RNase-free water/NTC, and the number of positive droplets in the FAM (Ch1) channel is 2 or more positive for methylation; the VIC (Ch2) channel is positive The number of droplets of 2 or more is judged as non-methylation positive, and the test results of the quality control products are shown in Figure 14.

5. Test results and analysis

The invention detects a total of 40 CRC patients and 13 healthy controls plasma free DNA, and calculates the number of methylation positive droplets and the number of non-methylation positive droplets for each sample according to the threshold line. The number of methylation positive droplets is in The proportion of the total number of positive droplets was taken as the methylation level of the sample. Both ZNF304 and LIFR in the plasma of colorectal cancer patients showed a hypermethylated state, as shown in Figure 15 and Figure 16 . The ROC curve was used to calculate the best cut-off values of the two genes, which were 0.003 and 0.002, respectively. The two genes and their combined positive rates were calculated in different stages. The results are shown in Table 16, Table 17, and Table 18.

Table 16

CRC patients ZNF304 positive/tested number Positive rate (%) Phase I 1/2 50% Phase II 5/9 55.6% Phase III 11/19 57.9% Stage IV 7/10 70% all patients 24/40 60% healthy control 1/13 7.7% (specificity 92.3%)

Table 17

Table 18

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<213> artificial sequence

<400> 25

caaaaaaacc aaaacgcgaa 20

<210> 26

<211> 13

<212> DNA

<213> artificial sequence

<400> 26

acgacgcctc gac 13

<210> 27

<211> 20

<212> DNA

<213> artificial sequence

<400> 27

tgattgttgg gattttgtga 20

<210> 28

<211> 21

<212> DNA

<213> artificial sequence

<400> 28

acccaacaaa aaaaccaaaa c 21

<210> 29

<211> 14

<212> DNA

<213> artificial sequence

<400> 29

caacacctca accc 14

<210> 30

<211> 69

<212> DNA

<213> artificial sequence

<400> 30

gttgtaggggg cgagatttttt ggcgtcgtcg tcgtgacgta ttttttttat gttcggttcg 60

tgtattttg 69

<210> 31

<211> 84

<212> DNA

<213> artificial sequence

<400> 31

gttgttttgg gttgtaggggg tgagattttt ggtgttgttg ttgtgatgta ttttttttat 60

gtttggtttg tgtattttgg ttgt 84

<210> 32

<211> 66

<212> DNA

<213> artificial sequence

<400> 32

gggattttgc gaattattta aatagtttag ggtcgaggcg tcgtcgttcg cgttttggtt 60

tttttg 66

<210> 33

<211> 80

<212> DNA

<213> artificial sequence

<400> 33

tgattgttgg gattttgtga attatttaaa tagtttaggg ttgaggtgtt gttgtttgtg 60

ttttggtttt ttttgttgggt 80

Claims (5)

1. Colorectal cancer-related methylation markers, wherein the methylation markers are any combination of ZNF304 containing CpG methylation sites and LIFR gene sequences or fragments thereof; According to the genome annotation information of the 450K chip, there is one CpG site on the LIFR gene, cg11841722, located at nucleotide 38557253 of the reverse strand of chromosome 5; There are two CpG sites in ZNF304 gene, cg10157975 and cg23250910, cg10157975 is located at the 57862442 nucleotide of the positive strand of chromosome 19, and cg23250910 is located at the 57862410 nucleotide of the anti-strand of chromosome 19. 2. The colorectal cancer-related methylation marker according to claim 1, wherein the methylation marker further comprises a C20orf194 gene sequence or a fragment thereof containing a CpG methylation site; According to the genome annotation information of the 450K chip, there is one CpG site on the C20orf194 gene, cg02893482, located at the 3388900th nucleotide of the positive strand of chromosome 20. 3. The primer set for amplifying the colorectal cancer-related methylation marker of claim 2, wherein the primer set is respectively used to amplify ZNF304 containing the CpG site, The primer pairs of LIFR and C20orf194 gene fragments are composed, and the primer sequences are as follows: ZNF304_2 F:5'-AYGTTAGAAGTTTYGTTTTGTAGTTTAGAG-3' R: 5'-AAATACATACCCTTTAAAAAACAACTATAACC-3' C20orf194F:5'-GATTTTAATAGTAAAGGTTAGGGGTTTT-3' R: 5'-CCAACRCACCTCCCCAATAC-3' LIFR F: 5’-GAYGGTTTTGYGGGGAGGA-3’ R: 5'-CCCCACACCCRACAAAAAA-3'. 4. A digital PCR test kit for colorectal cancer-related gene methylation detection, wherein the test kit comprises ddPCR primers for colorectal cancer-related gene ZNF304 and LIFR gene methylation site detection Probe combination, according to the genome annotation information of the 450K chip, there is one CpG site on the LIFR gene, cg11841722, which is located at the 38557253 nucleotide of the reverse strand of chromosome 5; there are two in the ZNF304 gene. CpG sites are cg13788592 and cg21627760 respectively, cg13788592 is located at nucleotide 57862612 of the reverse strand of chromosome 19, and cg21627760 is located at nucleotide 57862638 of the positive strand of chromosome 19; the ddPCR primer probe combination It consists of 1) and 2) as follows: 1) ddPCR primers and probes for ZNF304 methylation detection and ddPCR primers and probes for ZNF304 non-methylation detection, described primers and probe sequences are as follows: ZNF304 methylation forward primer 5'-GTTGTAGGGGCGAGATTT-3' ZNF304 methylation reverse primer 5'-CAAAATACACGAACCGAACATA-3' ZNF304 methylation probe 5’-TCACGACGACGACG-3’ ZNF304 unmethylated forward primer 5’-GTTGTTTTGGGTTGTAGGG-3’ ZNF304 Unmethylated Reverse Primer 5'-ACAACCAAAATACACAAACCA-3' ZNF304 unmethylated probe 5’-CATCACAACAACAACA-3’ 2) ddPCR primers and probes for LIFR methylation detection and PCR primers and probes for LIFR non-methylation detection, described primers and probe sequences are as follows: LIFR methylation forward primer 5'-GGGATTTTGCGAATTATTTAAATAG-3' LIFR methylation reverse primer 5'-CAAAAAAACCAAAACGCGAA-3' LIFR methylation probe 5'-ACGACGCCTCGAC-3' LIFR unmethylated forward primer 5'-TGATTGTTGGGATTTTGTGA-3' LIFR unmethylated reverse primer 5'-ACCCAACAAAAAAACCAAAAC-3' LIFR unmethylated probe 5'-CAACACCTCAACCC-3'. 5. The digital PCR kit of claim 4, wherein the 5' end of the probe comprises a fluorescent reporter group, and the fluorescent reporter group comprises FAM, HEX, NED, ROX, TET, Any one of JOE, TAMRA, CY3, CY5, and the methylated probe and the non-methylated probe use different fluorescent groups, and the 3' end of the probe contains a fluorescence quenching group, so The fluorescence quenching group includes any one of MGB, BHQ-1, BHQ-2 and BHQ-3.

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