Three-differential methylation marker composition for detecting liver cancer / early-stage liver cancer and application of three-differential methylation marker composition

Abstract

The invention provides a three-differential methylation marker composition for detecting liver cancer / early-stage liver cancer and application of the three-differential methylation marker composition, and belongs to the technical field of biological detection. The kit comprises three DNA methylation markers, and the composition of the three markers is significantly related to the occurrence of liver cancer. A combination result of methylation levels of the three markers is suitable for assisting doctors to judge whether early liver cancer occurs or not. According to the kit, more than 70% of samples for developing and using the reagent are samples (stage I or stage II) of early liver cancer, so that the discovery proportion of the early liver cancer in people with high risk of liver cancer in China is expected to be improved, early discovery and early treatment are realized, the morbidity and mortality of primary hepatocellular carcinoma are reduced, and the personal and social medical insurance pressure in society is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and specifically relates to a three-differential methylation marker composition for detecting liver cancer / early liver cancer and an application thereof. Background Art

[0002] The trend of liver cancer is similar to that of other cancers. In recent years, the incidence and mortality rates have tended to develop in younger age groups. The symptoms of liver cancer are not obvious in the early stage. If symptoms appear, the disease may have reached the late stage. Therefore, the prevention and treatment of liver cancer is particularly important.

[0003] At present, the common method of screening liver cancer in my country is liver ultrasound combined with serum alpha-fetoprotein (AFP) examination. Among them, ultrasound detection has the advantages of simple operation, non-invasiveness, and repeatability, but it is affected by the operator's experience level and the performance of the ultrasound equipment. It has high requirements for the size of the lesion, low sensitivity for monitoring small liver cancer, and most of them cannot determine the nature of liver nodules. AFP, as a specific tumor marker for liver cancer, has a sensitivity of only 40%~60%. Therefore, the monitoring method of abdominal ultrasound + AFP has obvious deficiencies in the screening of early liver cancer. It is urgent to develop more effective early screening and early diagnosis technologies to make up for the defects of existing monitoring methods and change the severe situation of liver cancer diagnosis and treatment in my country.

[0004] Abnormal DNA methylation is an early event in cancer development. Existing technologies have shown that circulating tumor DNA (Ct DNA) in blood-free DNA (cfDNA) is highly consistent with DNA in tumor tissue, indicating that early non-invasive detection of liver cancer can be achieved by detecting the methylation of blood cfDNA.

[0005] Therefore, product development through cfDNA methylation detection technology can detect early liver cancer with high sensitivity and good specificity, and can provide additional or even better options for clinical diagnosis. Summary of the invention

[0006] In view of the above-mentioned problem that liver cancer detection, especially early liver cancer detection technology is lacking and the sensitivity is not high, the purpose of the present invention is to provide a three-differentiated methylation marker composition for detecting liver cancer / early liver cancer, comprising three DNA methylation markers, and the three-marker composition is significantly related to liver cancer occurrence. The combined results of the methylation levels of the three markers are suitable for assisting doctors in making judgments on whether early liver cancer has occurred.

[0007] A three-differential methylation marker composition for detecting liver cancer / early liver cancer, the three-differential methylation marker composition comprising a methylation marker for detecting the methylation level of one or more of the following regions: chr5:42992299-42992764, chr14:53955979-53956219 and chr2:63054088-63054326; The genomic locations of the three differential methylation marker compositions are all determined based on the alignment of the human whole genome sequence hg38.

[0008] Preferably, the methylation marker chr5:42992299-42992764 is selected from the whole region or part of the region containing at least one CpG site as follows: region 1: chr5:42992299-42992414, region 2: chr5:42992451-42992557, region 3: chr5:42992522-42992764 and region 4: chr5:42992565-42992764; The methylation marker chr14:53955979-53956219 is selected from the whole region or a part of the region containing at least one CpG site: region 5: chr14:53955979-53956053, region 6: chr14:53956044-53956128 and region 7: chr14:53956135-53956219; The methylation marker chr2:63054088-63054326 is selected from the whole region or a part of the region containing at least one CpG site: region 8: chr2:63054088-63054212, region 9: chr2:63054112-63054305 and region 10: chr2:63054194-63054326.

[0009] Preferably, the combination of methylation markers consists of all or part of the 10 methylation regions shown as region 1 to region 10 in claim 2.

[0010] Application of a three-differentiated methylation marker composition for detecting liver cancer / early liver cancer in the preparation of a product for detecting liver cancer / early liver cancer.

[0011] A triple-differential methylation kit for detecting liver cancer / early liver cancer, the kit comprising the triple-differential methylation marker composition for detecting liver cancer / early liver cancer.

[0012] A nucleic acid composition for detecting liver cancer / early liver cancer, the nucleic acid composition comprising one or more of the following detection nucleic acid combinations: The nucleic acid combination 1 for detecting the methylation level in region 1 comprises upstream and downstream primers, nucleotide sequences such as SEQ ID NO.1-2, and a detection probe, nucleotide sequence such as SEQ ID NO.3; The nucleic acid combination 2 for detecting the methylation level in region 2 comprises upstream and downstream primers, the nucleotide sequences of which are SEQ ID NO.4-5, and a detection probe, the nucleotide sequence of which is SEQ ID NO.6; The nucleic acid combination 3 for detecting the methylation level in region 3 comprises upstream and downstream primers, the nucleotide sequences of which are SEQ ID NO.7-8, and a detection probe, the nucleotide sequence of which is SEQ ID NO.9; The nucleic acid combination 4 for detecting the methylation level in region 4 includes upstream and downstream primers, nucleotide sequences such as SEQ ID NO.10-11, and a detection probe, nucleotide sequence such as SEQ ID NO.12; The nucleic acid combination 5 for detecting the methylation level in region 5 comprises upstream and downstream primers, the nucleotide sequences of which are SEQ ID NO.13-14, and a detection probe, the nucleotide sequence of which is SEQ ID NO.15; The nucleic acid combination 6 for detecting the methylation level in region 6 comprises upstream and downstream primers, the nucleotide sequences of which are SEQ ID NO.16-17, and a detection probe, the nucleotide sequence of which is SEQ ID NO.18; The nucleic acid combination 7 for detecting the methylation level in region 7 comprises upstream and downstream primers, the nucleotide sequences of which are SEQ ID NO.19-20, and a detection probe, the nucleotide sequence of which is SEQ ID NO.21; The nucleic acid combination 8 for detecting the methylation level in region 8 comprises upstream and downstream primers, the nucleotide sequences of which are SEQ ID NO.22-23, and a detection probe, the nucleotide sequence of which is SEQ ID NO.24; The nucleic acid combination 9 for detecting the methylation level in region 9 includes upstream and downstream primers, nucleotide sequences such as SEQ ID NO.25-26, and a detection probe, nucleotide sequence such as SEQ ID NO.27; The nucleic acid assembly 10 for detecting the methylation level in the region 10 comprises upstream and downstream primers, nucleotide sequences such as SEQ ID NO.28-29, and a detection probe, nucleotide sequence such as SEQ ID NO.30.

[0013] A nucleic acid product for detecting liver cancer / early liver cancer, the nucleic acid product comprising the nucleic acid composition.

[0014] Preferably, the nucleic acid product comprises a sequence having at least 90% to 95% similarity to the base sequence shown in the nucleotide sequence 1-30 in the above-mentioned nucleic acid combination 1-10.

[0015] In the present application, the primer probe is about 20bp. 90% similarity means tolerating the difference of 2 bases, and 95% similarity means tolerating the difference of 1 base. For PCR reaction, the combination of primers also has a certain error tolerance, and it is not necessary for all bases to match. In addition, DNA comparison tools such as Blast can also usually tolerate certain base differences. What is detected in the present application is the DNA template converted by methylation, and most of C becomes T, so the complexity of the DNA sequence is reduced, and the error tolerance for DNA matching is relatively higher.

[0016] A nucleic acid kit for detecting liver cancer / early liver cancer, comprising the nucleic acid composition, dNTPs, Mg2+, methylation-sensitive restriction endonuclease and PCR buffer.

[0017] Preferably, the liver cancer includes primary liver cancer or intrahepatic cholangiocarcinoma or mixed hepatocellular carcinoma-intrahepatic cholangiocarcinoma.

[0018] Compared with the prior art, the present invention has at least the following technical effects: (I) The present invention provides a kit for detecting liver cancer / early liver cancer, the kit comprising three DNA methylation markers, the three-marker combination being significantly associated with liver cancer occurrence. The combined result of the methylation levels of the three markers is suitable for assisting doctors in making a judgment on whether early liver cancer has occurred.

[0019] (II) The kit uses DNA methylation markers. Compared with traditional tumor markers or liver cancer detection methods, DNA methylation markers have high detection sensitivity, more stable test results, and are more likely to be detected in the early stages of cancer development, which is conducive to the discovery of liver cancer in the early stages of development. The kit uses a combination of trimethylation markers, which can further improve the detection accuracy on the basis of a single methylation marker and avoid the problems of false detection and missed detection.

[0020] (III) The DNA methylation markers and compositions used in the kit are screened from genomic regions with a coverage rate 10 times that of a 450K methylation chip. In the sample data set used in the present invention, the detection performance of the screened markers and compositions is optimal.

[0021] (IV) The methylation sequencing used in the marker screening process is based on the enzymatic conversion method to convert cytosine to uracil. Compared with the traditional bisulfite conversion, it can avoid damage to DNA, thus solving the problem of low free DNA content in the blood.

[0022] (V) More than 70% of the samples used in the development of this kit are early liver cancer samples (stage I or II), which is expected to increase the detection rate of early liver cancer in high-risk populations in my country, achieve early detection and early treatment, reduce the incidence and mortality of primary hepatocellular carcinoma, and reduce the pressure on individuals and social medical insurance. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a flow chart of liver cancer methylation marker screening in Example 1; Figure 2 This is the ROC diagram of the detection of the methylation marker composition in Example 3 in an ex vivo blood sample of liver cancer. DETAILED DESCRIPTION

[0024] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0025] Example 1: Product methylation site screening process This embodiment discloses a methylation marker composition for detecting early liver cancer and a screening process thereof.

[0026] In order to mine and screen methylation markers with excellent detection performance, this embodiment first uses a customized hybrid capture large panel for targeted methylation sequencing. The hybrid capture panel covers 3.98 million CpG methylation sites in the human genome, including most of the CpG island regions and genes related to cancer occurrence, with a coverage range 10 times that of traditional DNA methylation chips. By performing targeted sequencing on 82 clinical frozen tissue samples and 115 ex vivo blood samples and comparing the changes in methylation patterns of cancer groups and non-cancer groups, this embodiment preliminarily identified 30,008 methylation sites that can clearly distinguish cancer samples from non-cancer samples in both tissue samples and blood samples as potential cancer markers.

[0027] Furthermore, a hybrid capture panel was designed based on the 30,008 methylation sites initially screened above for a second round of targeted methylation sequencing. This round of sequencing adopted a deep sequencing strategy, with an average sequencing depth of 500X, which significantly improved the accuracy of methylation level calculations and facilitated the accurate screening of markers in the second round. Targeted methylation sequencing was performed on ex vivo plasma samples from 218 clinical liver cancer patients and 196 ex vivo plasma samples from non-cancer groups, and univariate statistical analysis and machine learning methods were used to accurately screen out the DNA methylation markers with the best liver cancer detection performance and select the most effective ones.

[0028] Finally, primer and probe nucleic acid combinations of the qPCR detection platform were designed for the genomic regions of the screened methylation markers, and the performance of liver cancer detection was verified on the qPCR platform using ex vivo plasma samples from 32 clinical liver cancer patients and 47 ex vivo plasma samples from non-cancer groups.

[0029] Finally, the combination of trimethylation markers with the best performance for liver cancer detection was determined through univariate performance analysis and exhaustive combination methods, namely the methylation levels of the chr5:42992299-42992764, chr14:53955979-53956219 and chr2:63054088-63054326 genomic regions.

[0030] Example 2: Screening of significantly differentially methylated sites using the NGS platform NGS test steps: 1. Extraction of DNA samples: When the sample is a frozen fresh tissue sample, QIAGEN's "DNeasy® Blood & Tissue Kit #69506" is used to extract the genome. For specific operations, please refer to the kit instructions. Genomic DNA is interrupted using a Covaris M220 ultrasonic interrupter. When cfDNA is extracted from plasma samples, Guangzhou Youze Bio's "Free DNA Extraction Kit (Filtration Method) #C02-1" is used. For specific operations, please refer to the kit instructions.

[0031] 2. Use SPRIselect magnetic beads to screen the extracted DNA samples according to the recommended steps in the instructions, and filter out DNA molecules larger than 200 bp.

[0032] 3. Quality control preparation: 10 µL of 0.1 ng / µL CpG methylated pUC19 and 10 µL of 2 ng / µL unmethylated λDNA should be added to each sample; 4. Library construction for methylation sequencing: Use the NEBNext Enzymatic Methyl-Seq kit to construct the library.

[0033] 4.1 End repair and 3' end addition of "A" 4.1.1 Take 50 ng of the sample to be tested, dilute it to 50 μL with NF water, and then add the following reagents for reaction.

[0034]

[0035] 4.1.2 Place in PCR instrument and perform reaction according to the following procedure

[0036] 4.2 Connector connection 4.2.1 Add the following reagents to the end repair product for reaction

[0037] 4.2.2 Place in a PCR instrument and set the PCR reaction program according to the following conditions: 60°C, 60 min, with the heated lid closed.

[0038] 4.2.3 Purification of adapter ligation products A. Equilibrate the purified magnetic beads at room temperature for 30 minutes and mix them thoroughly on a vortex.

[0039] B. Prepare 80% ethanol according to the ratio of anhydrous ethanol to nuclease-free water = 8:2 and set aside.

[0040] C. Add 55 μL of purified magnetic beads to each ligation product, mix well by pipetting, incubate at room temperature for 5 minutes, place on a magnetic rack, and discard the supernatant after the solution is clarified; wash with freshly prepared 80% ethanol, discard the supernatant, and repeat this step once; dry, elute with 15 μL of elution buffer, and aspirate 14 μL of supernatant for the next reaction.

[0041] 4.3 Methylation: Oxidation reaction 4.3.1 Buffer configuration: a. Add 400 μL of E1 reaction buffer to 100 μL of E1 reaction buffer supplement, shake and mix thoroughly, and mark the preparation date.

[0042] b Take 10 μL of 500 mM Fe (II) and add it to 1249 μL of nuclease-free water. The diluted solution should be used immediately and should not be stored.

[0043] c Dilute the Stop Buffer 1:10 with nuclease-free water.

[0044] 4.3.2 Add the following reagents to 14 μL of the purified ligation product and mix well. Then add 10 μL of diluted Fe(II) to the purified ligation product to which the oxidase has been added, mix well and centrifuge.

[0045]

[0046] 4.3.3 Place the tube in a PCR instrument and react under the following conditions: 37°C, 1h, heating cover temperature ≥ 45°C.

[0047] 4.3.4 After the reaction is completed, add 1 μL of the diluted reaction stop solution to the product and place it on the PCR instrument for reaction under the following conditions: 37°C, 30 min, hot cover temperature ≥ 45°C.

[0048] 4.3.5 Purification of oxidation reaction products A. Equilibrate the purified magnetic beads at room temperature for 30 minutes and thoroughly mix them on a vortex machine; B. Prepare 80% ethanol in a ratio of 8:2 for absolute ethanol and nuclease-free water, and set aside. C. Add 45 μL of NEB Next Sample purified magnetic beads to each ligation product, mix well by pipetting, incubate at room temperature for 5 minutes, place on a magnetic rack, and discard the supernatant after the solution is clarified; wash with freshly prepared 80% ethanol, discard the supernatant, and repeat this step once; dry, elute with 9.5 μL of elution buffer, and aspirate 8 μL of supernatant for the next reaction.

[0049] 4.4 Methylation: cytosine deamination 4.4.1 Denaturation: a. Preheat the PCR instrument to 85°C and open the heated cover; b Add 2 μL of formamide to 8 μL of the purified oxidation reaction product, vortex to mix, and centrifuge briefly.

[0050] 4.4.2 Add the following reagents to the denatured product for reaction

[0051] 4.4.3 Place in PCR instrument and react under the following conditions: 37℃, 3h, heating cover temperature ≥ 45℃.

[0052] 4.4.4 Purification of Cytosine Deamination Reaction Products a. Equilibrate the purified magnetic beads at room temperature for 30 minutes and thoroughly mix them on a vortex machine; b Prepare 80% ethanol according to the ratio of anhydrous ethanol to nuclease-free water = 8:2 and set aside; c. Add 50 μL of NEB Next Sample purified magnetic beads to each ligation product, mix well by pipetting, incubate at room temperature for 5 minutes, place on a magnetic rack, and discard the supernatant after the solution is clarified; wash with freshly prepared 80% ethanol, discard the supernatant, and repeat this step once; dry, elute with 11 μL of elution buffer, and aspirate 10 μL of supernatant for the next reaction.

[0053] 4.5 PCR amplification and purification 4.5.1 Add the following reagents to the above purified product for reaction

[0054] 4.5.2 Place on PCR instrument and react according to the following conditions

[0055] 4.5.3 Purification of PCR amplification products a. Equilibrate the purified magnetic beads at room temperature for 30 minutes and thoroughly mix them on a vortex machine; b Prepare 80% ethanol in the ratio of anhydrous ethanol to nuclease-free water = 8:2 and set aside; c. Add 22.5 μL of NEB purified magnetic beads to each ligation product, mix well by pipetting, incubate at room temperature for 5 minutes, place on a magnetic rack, and discard the supernatant after the solution is clarified; wash with freshly prepared 80% ethanol, discard the supernatant, and repeat this step once; dry, elute with 15 μL of elution buffer, aspirate 14 μL of supernatant, and conduct quality inspection.

[0056] 5. Hybridization and elution 5.1 Hybridization: a Take 10 ng of each library to be hybridized, calculate the library volume required for combined hybridization, and mix the 12 libraries together; b. Add the following reagents to the library mixture for reaction; c Turn on the vacuum concentrator, set the V-AQ mode, and concentrate into dry powder at room temperature; d. Immediately take 20 μL of hybridization mixture and add it to the concentrated dry powder, mix well and let it stand at room temperature for 5 minutes, then add 30 μL of hybridization enhancement solution, mix well and centrifuge; e Place on a PCR instrument and perform the reaction under the following conditions.

[0057] Reagent composition table

[0058] PCR reaction conditions table

[0059] 5.2 Capture and elution 5.2.1 Buffer preheating: Preheat the rapid binding solution and rapid washing solution 2 at 48°C until the precipitate is dissolved, and preheat the rapid washing solution 1 at 63°C until the precipitate is dissolved. Equilibrate the streptavidin magnetic beads at room temperature for at least 30 minutes.

[0060] 5.2.2 Magnetic bead capture a Vortex to mix the streptavidin magnetic beads that have been equilibrated to room temperature; b. Take 70 μL of streptavidin magnetic beads and add them into a 1.5 mL centrifuge tube; c. Add 20 μL of binding buffer, mix by pipetting, centrifuge briefly, place on a magnetic stand, let stand for 1 min, and discard the supernatant; d. Repeat steps b and c twice, performing streptavidin magnetic bead washing three times in total; e Add 20 μL binding buffer to resuspend the streptavidin magnetic beads; f Transfer 20 μL of resuspended streptavidin magnetic beads to the hybridization reaction tube, and then transfer all the liquid to a 1.5 mL centrifuge tube.

[0061] g. Mix on a mixer at room temperature for 30 minutes without vortexing. h After mixing, centrifuge immediately, place on a magnetic stand, let stand for 1 minute, and discard the supernatant.

[0062] 5.2.3 Elution a. Add 200 μL of 54°C preheated rapid wash buffer 1, mix well by pipetting, and incubate in a thermostatic metal bath at 54°C for 5 min; b After the incubation is completed, centrifuge briefly and transfer all the liquid to a new 1.5 mL centrifuge tube to remove the non-specific capture fragments bound to the surface of the centrifuge tube; c Place on a magnetic stand, let stand for 1 minute, and discard the supernatant; d. Add 200 μL of 63°C preheated washing buffer 1, pipette to mix, and incubate in a thermostatic metal bath at 54°C for 5 min. e After incubation, centrifuge immediately, place on a magnetic stand, let stand for 1 min, and discard the supernatant; f. Add 200 μL of 54°C preheated washing buffer 2, pipette to mix, and incubate in a thermostatic metal bath at 54°C for 5 minutes; g After the incubation is completed, centrifuge briefly, place on a magnetic stand, let stand for 1 minute, and discard the supernatant; h. Repeat steps a to g twice for a total of three washes; i Centrifuge briefly, discard the remaining supernatant with a 10 μL pipette tip, immediately add 45 μL of nuclease-free water, pipette to mix, and incubate on ice.

[0063] 5.3 PCR enrichment of capture products 5.3.1 Take 22.5 μL of the captured product (streptavidin magnetic bead suspension) and add the following reagents to react.

[0064]

[0065] Place on PCR instrument and react according to the following conditions

[0066] 5.3.2 PCR product purification a. Equilibrate the DNA purification magnetic beads at room temperature for 30 minutes and mix thoroughly on a vortex machine; b Prepare 80% ethanol in the ratio of anhydrous ethanol to nuclease-free water = 8:2 and set aside; c. Add 90 μL of DNA purification magnetic beads to each ligation product, mix well by pipetting, incubate at room temperature for 5 minutes, place on a magnetic rack, discard the supernatant after the solution is clarified; wash with freshly prepared 80% ethanol, discard the supernatant, repeat this step once; dry, elute with 32 μL of elution buffer, aspirate 30 μL of supernatant for quality control, and sequence on the machine.

[0067] 6. Analysis of sequencing data: The raw data was first filtered using fastQC to filter low-quality sequences (phred33 score ≤ 20) and adapter sequences and polyA / T sequences in the reads. The filtered high-quality reads were mapped to the hg38 human genome using BSMAP, and reads with high mapping quality were screened out. After removing PCR duplicates using picard, reads mapped to the target genomic region (the region corresponding to the DNA methylation marker) were selected using samtools and the methylation levels of each DNA methylation marker were calculated.

[0068] The screening of methylation markers on the NGS platform is divided into two steps. The first step is to use a customized large methylation panel for preliminary site screening. The samples used in this step are shown in Table 1 below:

[0069] Based on the data results of the first step tissue sequencing analysis, a customized capture probe panel with a size of 480K was designed.

[0070] Example 3: NGS liver cancer ex vivo plasma samples screen for optimal sites on a 480K panel The NGS test steps are consistent with the NGS test steps in Example 2.

[0071] Based on the 480K panel, we further screened for differentially methylated sites. NGS sequencing analysis was performed based on the 480K methylation panel, and the sample details are shown in the following table:

[0072] After screening the results of 480K hybridization capture panel NGS sequencing, we screened and obtained the following gene regions that can be used as candidate markers for subsequent qPCR.

[0073] The sensitivity and specificity of the candidate regions in liver cancer ex vivo plasma samples are shown in the following table:

[0074] like Figure 2 As shown, the AUC value of the determined three-marker methylation combination for methylation sequencing detection of liver cancer ex vivo blood samples is 0.955.

[0075] Example 4: qPCR platform validation of candidate site performance In order to further verify the performance of the selected gene region, we used the qPCR platform to verify the selected region. The sample details of this example are shown in the following table:

[0076] This example uses a methylation-specific PCR method to detect the methylation level of genes in plasma samples of liver cancer in vitro, and the specific steps are as follows: 1. Separation of plasma: First, centrifuge the collected 10 mL clinical blood sample at 1600×g for 10 minutes to separate the plasma. Centrifuge the separated plasma at 16000×g for 10 minutes to separate impurities. The purified plasma sample should be stored in a -80℃ refrigerator for future use.

[0077] 2. Free DNA extraction: Use the "Free DNA Extraction Kit" (Cat. No.: C03-2) of Guangzhou Youze Biotechnology Co., Ltd. to extract plasma free DNA from the separated plasma samples.

[0078] 3. Free DNA conversion: Use ZYMO's "EZ-96 DNA Methvlation-Lightning MagPrep" to perform methylation C->T conversion on the extracted free DNA samples.

[0079] 4. Negative and positive controls for methylation quantitative PCR: The negative control was the genomic DNA of the cell line (cell line: QSG-7701) transformed by the ZYMO kit, with a concentration of 1 ng / μL. After transformation, the C in the negative control was changed to T. The positive control was the genomic DNA of the cell line (cell line: HepG2) transformed by bisulfite using the ZYMO kit, with a concentration of 1 ng / μL. After transformation, the C at the methylated CpG position did not change, and the C at other positions was changed to T.

[0080] 5. Reaction system for methylation quantitative PCR:

[0081] 6. Reaction procedure of methylation quantitative PCR:

[0082] 7. On-machine detection: Mix the sample DNA to be tested, negative reference, and positive reference according to the reaction system of methylation quantitative PCR in step 5. Put the sample into Shanghai Hongshi SLAN-96S quantitative PCR instrument, and set up the quantitative PCR instrument according to the program conditions in step 6 for detection, and collect the fluorescence values ​​of each channel of HEX / ROX / FAM / CY5.

[0083] 8. Result recording: After the methylation quantitative PCR reaction is completed, the threshold is set at the inflection point of the S-type amplification curve to obtain the Ct value of the sample to be tested at the methylation site.

[0084] 9. Quality control: Negative and positive references are tested simultaneously in each test. The negative control is the transformed cell genomic DNA (cell line: QSG-7701) containing the β-actin gene and the low-methylation target gene sequence, and the positive control is the transformed cell cfDNA (cell line: HepG2) containing the β-actin gene and the high-methylation target gene sequence, both at a concentration of 1ng / μL. When the negative reference is not amplified, the Ct value of the positive reference is between 28-32, and the Ct value of the internal reference gene is less than 38, the experiment is valid and the next step of sample result determination can be carried out. Otherwise, the result is invalid and the test must be repeated.

[0085] 10. Result determination: If the Ct value of the target marker is less than 35, the test result is positive, otherwise the test result is negative. When multiple markers are combined for joint determination, if at least one target marker's Ct value is less than 35, the test result is positive, otherwise the test result is negative.

[0086] This embodiment also provides a diagnostic reagent for primary hepatocellular carcinoma, which includes the following nucleic acid combination: Nucleic acid combination 1 includes primer pair 1 and probe 1. The primer pair includes upstream primer sequence 1, downstream primer sequence 2 and probe 1 sequence 3, and is used to amplify the fragment between region 1.

[0087] The nucleic acid combination 2 includes a primer pair 2 and a probe 2 , wherein the primers include an upstream primer sequence 4 , a downstream primer sequence 5 , and a probe 2 sequence 6 , and are used to amplify a fragment between regions 2 .

[0088] The nucleic acid combination 3 includes a primer pair 3 and a probe 3 . The primer pair includes an upstream primer sequence 7 , a downstream primer sequence 8 , and a probe 3 sequence 9 , and is used to amplify the fragment between the regions 3 .

[0089] The nucleic acid combination 4 includes a primer pair 4 and a probe 4 . The primer pair includes an upstream primer sequence 10 , a downstream primer sequence 11 and a probe sequence 12 , and is used to amplify the fragment between the regions 4 . The nucleic acid combination 5 includes a primer pair 5 and a probe 5 . The primer pair includes an upstream primer sequence 13 , a downstream primer sequence 14 , and a probe 5 sequence 15 , and is used to amplify the fragment between the regions 5 .

[0090] The nucleic acid combination 6 includes a primer pair 6 and a probe 6 . The primer pair includes an upstream primer sequence 16 , a downstream primer sequence 17 , and a probe 6 sequence 18 , and is used to amplify the fragment between the regions 6 . The nucleic acid combination 7 includes a primer pair 7 and a probe 7 . The primer pair includes an upstream primer sequence 19 , a downstream primer sequence 20 , and a probe 7 sequence 21 , and is used to amplify the fragment between the regions 7 . The nucleic acid combination 8 includes a primer pair 8 and a probe 8 . The primer pair includes an upstream primer sequence 22 , a downstream primer sequence 23 , and a probe 8 sequence 24 , and is used to amplify the fragment between the regions 8 . The nucleic acid combination 9 includes a primer pair 9 and a probe 9 . The primer pair includes an upstream primer sequence 25 , a downstream primer sequence 26 and a probe sequence 27 , and is used to amplify the fragment between the regions 9 .

[0091] The nucleic acid combination 10 includes a primer pair 10 and a probe 10 . The primer pair includes an upstream primer sequence 28 , a downstream primer sequence 29 and a probe 10 sequence 30 , and is used to amplify a fragment between regions 10 .

[0092] The detection performance results of different nucleic acid combinations on the qPCR detection platform for ex vivo liver cancer blood samples are shown in the following table:

[0093] From the results of plasma qPCR test, it can be seen that the detection sensitivity of ex vivo blood samples of liver cancer using chr5:42992299-42992764, chr14:53955979-53956219 and chr2:63054088-63054326 methylation markers is 78.1% to 87.5%, and the specificity is about 90%. When the three markers are combined, the detection sensitivity can be increased to 90.6-93.8%, and the specificity remains at about 90%.

[0094] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A three-differential methylation marker composition for detecting liver cancer / early liver cancer, characterized in that: The three differential methylation marker compositions include for detecting the methylation levels of one or more of the following regions: chr5:42992299-42992764, chr14:53955979-53956219 and chr2:63054088-63054326; The genomic locations of the three differential methylation marker compositions are all determined based on the alignment of the human whole genome sequence hg38.

2. A three-differential methylation marker composition for detecting liver cancer / early liver cancer according to claim 1, characterized in that: The methylation marker chr5:42992299-42992764 is selected from the whole region or part of the region containing at least one CpG site: region 1: chr5:42992299-42992414, region 2: chr5:42992451-42992557, region 3: chr5:42992522-42992764 and region 4: chr5:42992565-42992764; The methylation marker chr14:53955979-53956219 is selected from the whole region or a part of the region containing at least one CpG site: region 5: chr14:53955979-53956053, region 6: chr14:53956044-53956128 and region 7: chr14:53956135-53956219; The methylation marker chr2:63054088-63054326 is selected from the whole region or a part of the region containing at least one CpG site: region 8: chr2:63054088-63054212, region 9: chr2:63054112-63054305 and region 10: chr2:63054194-63054326.

3. A three-differential methylation marker composition for detecting liver cancer / early liver cancer according to claim 2, characterized in that: The combination of methylation markers consists of all or part of the 10 methylation regions shown as region 1 to region 10 in claim 2 .

4. Use of the three-differential methylation marker composition for detecting liver cancer / early liver cancer as described in any one of claims 1 to 3 in the preparation of a product for detecting liver cancer / early liver cancer.

5. A triple differential methylation kit for detecting liver cancer / early liver cancer, characterized in that: The kit comprises the three-differential methylation marker composition for detecting liver cancer / early liver cancer as described in any one of claims 1 to 3.

6. A nucleic acid composition for detecting liver cancer / early liver cancer, characterized in that: The nucleic acid composition includes one or more of the following detection nucleic acid combinations: The nucleic acid combination 1 for detecting the methylation level in region 1 as claimed in claim 2 comprises upstream and downstream primers, nucleotide sequences such as SEQ ID NO.1-2, and a detection probe, nucleotide sequence such as SEQ ID NO.3; The nucleic acid combination 2 for detecting the methylation level in region 2 comprises upstream and downstream primers, the nucleotide sequences of which are SEQ ID NO.4-5, and a detection probe, the nucleotide sequence of which is SEQ ID NO.6; The nucleic acid combination 3 for detecting the methylation level in region 3 comprises upstream and downstream primers, the nucleotide sequences of which are SEQ ID NO.7-8, and a detection probe, the nucleotide sequence of which is SEQ ID NO.9; The nucleic acid combination 4 for detecting the methylation level in region 4 includes upstream and downstream primers, nucleotide sequences such as SEQ ID NO.10-11, and a detection probe, nucleotide sequence such as SEQ ID NO.12; The nucleic acid combination 5 for detecting the methylation level in region 5 comprises upstream and downstream primers, the nucleotide sequences of which are SEQ ID NO.13-14, and a detection probe, the nucleotide sequence of which is SEQ ID NO.15; The nucleic acid combination 6 for detecting the methylation level in region 6 comprises upstream and downstream primers, the nucleotide sequences of which are SEQ ID NO.16-17, and a detection probe, the nucleotide sequence of which is SEQ ID NO.18; The nucleic acid combination 7 for detecting the methylation level in region 7 comprises upstream and downstream primers, the nucleotide sequences of which are SEQ ID NO.19-20, and a detection probe, the nucleotide sequence of which is SEQ ID NO.21; The nucleic acid combination 8 for detecting the methylation level in region 8 comprises upstream and downstream primers, the nucleotide sequences of which are SEQ ID NO.22-23, and a detection probe, the nucleotide sequence of which is SEQ ID NO.24; The nucleic acid combination 9 for detecting the methylation level in region 9 includes upstream and downstream primers, nucleotide sequences such as SEQ ID NO.25-26, and a detection probe, nucleotide sequence such as SEQ ID NO.27; The nucleic acid assembly 10 for detecting the methylation level in the region 10 comprises upstream and downstream primers, nucleotide sequences such as SEQ ID NO.28-29, and a detection probe, nucleotide sequence such as SEQ ID NO.

30.

7. A nucleic acid product for detecting liver cancer / early liver cancer, characterized in that: The nucleic acid product comprises the nucleic acid composition according to claim 6.

8. A nucleic acid product for detecting liver cancer / early liver cancer according to claim 7, characterized in that: The nucleic acid product comprises a sequence having at least 90% to 95% similarity to the base sequence shown in the nucleotide sequence 1-30 in the above-mentioned nucleic acid combination 1-10.

9. A nucleic acid kit for detecting liver cancer / early liver cancer, characterized in that: The nucleic acid kit comprises the nucleic acid composition as claimed in claim 6, dNTPs, Mg2+, a methylation-sensitive restriction endonuclease and a PCR buffer.

10. A nucleic acid kit for detecting liver cancer / early liver cancer according to claim 9, characterized in that: The liver cancer includes primary liver cancer or intrahepatic cholangiocarcinoma or mixed hepatocellular carcinoma-intrahepatic cholangiocarcinoma.

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