Three-differentiated methylation marker composition for detecting liver cancer / early liver cancer and application thereof

Abstract

The application provides a three-differentiated methylation marker composition for detecting liver cancer / early liver cancer and application thereof, and belongs to the technical field of biological detection. The kit contains three DNA methylation markers, and the three-marker composition is significantly related to liver cancer. Through the combined results of the methylation levels of the three markers, the doctor can make a judgment on whether early liver cancer occurs. More than 70% of the samples used in the development of the kit reagent are early liver cancer samples (stage I or stage II), which is expected to improve the discovery rate of early liver cancer in the high-risk population of liver cancer in China, realize early discovery and early treatment, reduce the incidence and mortality of primary hepatocellular carcinoma, and reduce the pressure on individuals and social medical insurance.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a three-differential methylation biomarker composition for detecting liver cancer / early liver cancer and its application. Background Technology

[0002] Similar to other cancers, liver cancer has shown a trend of increasing incidence and mortality rates among younger age groups in recent years. Early-stage liver cancer often presents with few or no symptoms; by the time symptoms appear, the disease may already be in an advanced stage. Therefore, prevention and treatment of liver cancer are of paramount importance.

[0003] The common clinical method for screening liver cancer is liver ultrasound combined with serum alpha-fetoprotein (AFP) testing. Ultrasound has advantages such as simplicity, non-invasiveness, and repeatability; however, it is affected by the operator's experience and the performance of the ultrasound equipment, has high requirements regarding lesion size, and has low sensitivity for detecting small liver cancers, often failing to determine the nature of liver nodules. AFP, as a specific tumor marker for liver cancer, has a sensitivity of only 40%–60%. Therefore, the abdominal ultrasound + AFP monitoring method is significantly insufficient for early liver cancer screening, and there is an urgent need to develop more effective early screening and diagnosis technologies to compensate for the deficiencies of existing monitoring methods.

[0004] Abnormal DNA methylation is an early event in cancer development. Current technology shows that circulating tumor DNA (Ct DNA) in blood cell-free DNA (cfDNA) has a strong consistency 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 using cfDNA methylation detection technology can detect early-stage liver cancer with high sensitivity and specificity, providing additional or even better options for clinical diagnosis. Summary of the Invention

[0006] To address the aforementioned issues of insufficient and low-sensitivity technologies for liver cancer detection, especially early-stage liver cancer detection, the present invention aims to provide a three-differential methylation biomarker composition for detecting liver cancer / early-stage liver cancer. This composition comprises three DNA methylation biomarkers, and this three-biomarker composition is significantly correlated with the occurrence of liver cancer. The combined results of the methylation levels of these three biomarkers are suitable for assisting physicians in determining whether early-stage liver cancer has occurred.

[0007] A tri-differential methylation biomarker composition for detecting liver cancer / early-stage liver cancer, the tri-differential methylation biomarker composition comprising the following regions for detecting methylation levels: chr5:42992299-42992764, chr14:53955979-53956219 and chr2:63054088-63054326;

[0008] The genomic localization of the three differentially methylated biomarker compositions was determined based on the alignment of the human whole genome sequence with hg38.

[0009] Preferably, the methylation marker chr5:42992299-42992764 is selected from the whole or part of a region containing at least one CpG site from the following regions: region 1: chr5:42992299-42992414, region 2: chr5:42992451-42992557, region 3: chr5:42992522-42992764 and region 4: chr5:42992565-42992764;

[0010] The methylation marker chr14:53955979-53956219 is selected from the entire or partial regions of one of the following regions that contain at least one CpG site: region 5: chr14:53955979-53956053, region 6: chr14:53956044-53956128 and region 7: chr14:53956135-53956219;

[0011] The methylation marker chr2:63054088-63054326 is selected from the entire or partial regions of one of the following regions that contain at least one CpG site: region 8: chr2:63054088-63054212, region 9: chr2:63054112-63054305, and region 10: chr2:63054194-63054326.

[0012] Preferably, the combination of methylation markers consists of all or part of the 10 methylation regions shown in regions 1-10 of claim 2.

[0013] Application of a tri-differential methylation biomarker composition for detecting liver cancer / early-stage liver cancer in the preparation of products for detecting liver cancer / early-stage liver cancer.

[0014] A tri-differential methylation kit for detecting liver cancer / early-stage liver cancer, the kit comprising the aforementioned tri-differential methylation biomarker composition for detecting liver cancer / early-stage liver cancer.

[0015] A nucleic acid composition for detecting liver cancer / early-stage liver cancer, said nucleic acid composition comprising one or more of the following detection nucleic acid combinations:

[0016] Nucleic acid assembly 1 for detecting methylation levels in region 1 includes upstream and downstream primers, nucleotide sequences as shown in SEQ ID NO. 1-2, and a detection probe, nucleotide sequence as shown in SEQ ID NO. 3;

[0017] Nucleic acid assembly 2 for detecting methylation levels in region 2 includes upstream and downstream primers, nucleotide sequences as shown in SEQ ID NO. 4-5, and a detection probe, nucleotide sequence as shown in SEQ ID NO. 6;

[0018] Nucleic acid assembly 3 for detecting methylation levels in region 3 includes upstream and downstream primers, nucleotide sequences as shown in SEQ ID NO. 7-8, and a detection probe, nucleotide sequence as shown in SEQ ID NO. 9;

[0019] Nucleic acid assembly 4 for detecting methylation levels in region 4 includes upstream and downstream primers, nucleotide sequences as shown in SEQ ID NO. 10-11, and a detection probe, nucleotide sequence as shown in SEQ ID NO. 12;

[0020] Nucleic acid assembly 5 for detecting methylation levels in region 5 includes upstream and downstream primers, nucleotide sequences as shown in SEQ ID NO.13-14, and a detection probe, nucleotide sequence as shown in SEQ ID NO.15;

[0021] Nucleic acid combination 6 for detecting methylation levels in region 6 includes upstream and downstream primers, nucleotide sequences as shown in SEQ ID NO. 16-17, and a detection probe, nucleotide sequence as shown in SEQ ID NO. 18;

[0022] Nucleic acid assembly 7 for detecting methylation levels in region 7 includes upstream and downstream primers, nucleotide sequences as shown in SEQ ID NO. 19-20, and a detection probe, nucleotide sequence as shown in SEQ ID NO. 21;

[0023] Nucleic acid assembly 8 for detecting methylation levels in region 8 includes upstream and downstream primers, nucleotide sequences as shown in SEQ ID NO. 22-23, and a detection probe, nucleotide sequence as shown in SEQ ID NO. 24;

[0024] Nucleic acid assembly 9 for detecting methylation levels in region 9 includes upstream and downstream primers, nucleotide sequences as shown in SEQ ID NO. 25-26, and a detection probe, nucleotide sequence as shown in SEQ ID NO. 27;

[0025] Nucleic acid assembly 10 for detecting methylation levels in region 10 includes upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO. 28-29, and a detection probe with a nucleotide sequence as shown in SEQ ID NO. 30.

[0026] A nucleic acid product for detecting liver cancer / early-stage liver cancer, said nucleic acid product comprising the aforementioned nucleic acid composition.

[0027] Preferably, the nucleic acid product contains a sequence having at least 90% to 95% similarity to the base sequence shown in nucleotide sequences 1-30 of the above-described nucleic acid combinations 1-10.

[0028] In this application, the primers and probes are approximately 20 bp in length. 90% similarity means tolerating differences of two bases, and 95% similarity means tolerating differences of one base. For PCR reactions, primer binding also has a certain tolerance for error; not all bases need to match. Furthermore, DNA alignment tools such as BLAST typically tolerate a certain number of base differences. This application detects a methylated DNA template, where most Cs are replaced with Ts, thus reducing the complexity of the DNA sequence and resulting in a relatively higher tolerance for DNA matching errors.

[0029] A nucleic acid kit for detecting liver cancer / early-stage liver cancer, the nucleic acid kit comprising the aforementioned nucleic acid composition, dNTPs, Mg2+, methylation-sensitive restriction endonuclease, and PCR buffer.

[0030] Preferably, the liver cancer includes primary liver cancer, intrahepatic cholangiocarcinoma, or mixed hepatocellular carcinoma-intrahepatic cholangiocarcinoma.

[0031] Compared with the prior art, the present invention has at least the following technical effects:

[0032] (i) This invention provides a kit for detecting liver cancer / early-stage liver cancer, which contains three DNA methylation markers. The combination of these three markers is significantly associated with the occurrence of liver cancer. The combined results of the methylation levels of the three markers are suitable for assisting doctors in determining whether early-stage liver cancer has occurred.

[0033] (ii) This kit uses DNA methylation markers. Compared to traditional tumor markers or liver cancer detection methods, DNA methylation markers offer higher sensitivity, more stable results, and are more likely to be detected in the early stages of cancer development, thus facilitating the early detection of liver cancer. This kit uses a combination of trimethylation markers, which further improves detection accuracy based on single methylation markers, avoiding false positives and false negatives.

[0034] (iii) The DNA methylation markers and compositions used in this kit were screened from genomic regions with a coverage of 10 times that of the 450K methylation chip. Among the sample datasets used in this invention, the screened markers and compositions have the best detection performance.

[0035] (iv) The methylation sequencing used in the biomarker screening process is based on the enzymatic conversion method to convert cytosine to uracil. Compared with the traditional bisulfite conversion, it can avoid DNA damage, thereby solving the problem of low blood cell-free DNA content.

[0036] (v) More than 70% of the samples used in the development of this reagent kit were early-stage liver cancer samples (stage I or II), which is expected to increase the detection rate of early-stage liver cancer in high-risk groups and achieve early detection and early treatment. Attached Figure Description

[0037] Figure 1 This is a flowchart of the liver cancer methylation marker screening process in Example 1;

[0038] Figure 2 This is the ROC plot of the methylation marker composition in Example 3 in an ex vivo blood sample from liver cancer. Detailed Implementation

[0039] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0040] Example 1: Screening process for product methylation sites

[0041] This embodiment discloses a methylation biomarker composition for detecting early-stage liver cancer and the process for screening it.

[0042] To identify and screen methylation biomarkers with superior detection performance, this embodiment first employed a customized hybridization capture panel for targeted methylation sequencing. This hybridization capture panel covers 3.98 million CpG methylation sites in the human genome, encompassing the vast majority of CpG island regions and cancer-related genes, with a coverage area 10 times larger than that of traditional DNA methylation chips. By performing targeted sequencing on 82 clinical frozen tissue samples and 115 ex vivo blood samples and comparing changes in methylation patterns between cancer and non-cancer groups, this embodiment preliminarily identified 30,008 methylation sites in both tissue and blood samples that could clearly distinguish cancer and non-cancer samples as potential cancer biomarkers.

[0043] Furthermore, a hybridization capture panel was designed based on the 30,008 methylation sites initially screened for a second round of targeted methylation sequencing. This round of sequencing employed a deep sequencing strategy, achieving an average sequencing depth of 500X, significantly improving the accuracy of methylation level calculations and facilitating precise biomarker screening in the second round. Targeted methylation sequencing was performed on plasma samples from 218 clinical liver cancer patients and 196 non-cancer patients. Univariate statistical analysis and machine learning methods were used to accurately screen for DNA methylation biomarkers with the best detection performance for liver cancer, and the most effective biomarkers were selected from these.

[0044] Finally, primer and probe nucleic acid combinations for 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 in vitro plasma samples from 32 clinical liver cancer patients and 47 non-cancer patients.

[0045] Finally, through univariate performance analysis and exhaustive combination methods, the combination of trimethylation markers with the best detection performance for liver cancer was determined, namely the methylation levels of the genomic regions chr5:42992299-42992764, chr14:53955979-53956219, and chr2:63054088-63054326.

[0046] Example 2: Screening for significantly differentially methylated sites using an NGS platform

[0047] NGS test procedure:

[0048] 1. DNA Sample Extraction: When the sample is a frozen fresh tissue sample, the genomic DNA is extracted using QIAGEN's DNeasy® Blood & Tissue Kit #69506. Refer to the kit instructions for specific procedures. Genomic DNA is fragmented using a Covaris M220 ultrasonic fragmentation device. When extracting cfDNA from plasma samples, Guangzhou Youze Biotechnology's Cell-Free DNA Extraction Kit (Filtration Method) #C02-1 is used. Refer to the kit instructions for specific procedures.

[0049] 2. Use SPRIselect magnetic beads to screen the extracted DNA sample according to the steps recommended in the instruction manual, filtering out DNA molecules larger than 200bp.

[0050] 3. Quality control preparation: Add 10 µL of 0.1 ng / µL CpG-methylated pUC19 and 10 µL of 2 ng / µL unmethylated λDNA to each sample;

[0051] 4. Library construction for methylation sequencing: Library construction was performed using the NEBNext Enzymatic Methyl-Seq kit.

[0052] 4.1 End repair and 3' end addition of "A"

[0053] 4.1.1 Take 50 ng of the sample to be tested, dilute it with NF water to 50 μL, and then add the following reagents to carry out the reaction.

[0054]

[0055] 4.1.2 Place the sample in a PCR instrument and perform the reaction according to the following procedure.

[0056]

[0057] 4.2 Connector Connection

[0058] 4.2.1 Add the following reagents to the end-repair product to carry out the reaction.

[0059]

[0060] 4.2.2 Place it in a PCR instrument and set the PCR reaction program according to the following conditions: 60℃, 60min, with the hot lid closed.

[0061] 4.2.3 Purification of Connector Ligation Products

[0062] A. Equilibrate the purified magnetic beads at room temperature for 30 minutes, and then thoroughly mix the purified magnetic beads using a vortex mixer.

[0063] B. Prepare 80% ethanol by mixing anhydrous ethanol and nuclease-free water in a ratio of 8:2, and set aside.

[0064] C. Add 55 μL of purified magnetic beads to the ligation product and mix by pipetting. Incubate at room temperature for 5 minutes. Place on a magnetic rack and wait for the solution to become clear. Discard the supernatant. Wash with freshly prepared 80% ethanol and discard the supernatant. Repeat this step once. Air dry and elute with 15 μL of elution buffer. Take 14 μL of the supernatant for the next reaction.

[0065] 4.3 Methylation: Oxidation reaction

[0066] 4.3.1 Buffer configuration:

[0067] a. Add 400 μL of E1 reaction buffer to 100 μL of E1 reaction buffer replenishment solution, vortex to mix, and mark the preparation date.

[0068] b. Add 10 μL of 500 mM Fe(II) to 1249 μL of nuclease-free water. Use the diluted solution immediately and do not store it.

[0069] c. Dilute the stop buffer with nuclease-free water at a ratio of 1:10.

[0070] 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 containing oxidase and mix well before centrifugation.

[0071]

[0072] 4.3.3 Place it on a PCR instrument and react under the following conditions: 37℃, 1h, hot lid temperature ≥45℃.

[0073] 4.3.4 After the reaction is complete, add 1 μL of diluted reaction stop solution to the product and place it on a PCR instrument. React under the following conditions: 37℃, 30 min, and hot cap temperature ≥ 45℃.

[0074] 4.3.5 Purification of Oxidation Reaction Products

[0075] A. Equilibrate the purified magnetic beads at room temperature for 30 minutes, and then thoroughly mix the purified magnetic beads using a vortex mixer.

[0076] B. Prepare 80% ethanol by mixing anhydrous ethanol and nuclease-free water in a ratio of 8:2, and set aside.

[0077] C. Add 45 μL of NEB Next Sample purification magnetic beads to the ligation product, mix by pipetting, incubate at room temperature for 5 minutes, place on a magnetic rack, and discard the supernatant after the solution becomes clear; wash with freshly prepared 80% ethanol and discard the supernatant, repeat this step once; air dry, elute with 9.5 μL of elution buffer, and take 8 μL of supernatant for the next reaction.

[0078] 4.4 Methylation: Cytosine deamination

[0079] 4.4.1 Transgenderism:

[0080] Preheat the PCR instrument to 85°C and open the heated lid.

[0081] Add 2 μL of formamide to 8 μL of purified oxidation product, vortex to mix, and then centrifuge briefly.

[0082] 4.4.2 The following reagents were added to the denatured product to carry out the reaction.

[0083]

[0084] 4.4.3 Place it on a PCR instrument and react under the following conditions: 37℃, 3h, with the hot lid temperature ≥45℃.

[0085] 4.4.4 Purification of cytosine deamination product

[0086] a. Equilibrate the purified magnetic beads at room temperature for 30 minutes, and then thoroughly mix the purified magnetic beads using a vortex mixer;

[0087] b. Prepare 80% ethanol by mixing anhydrous ethanol and nuclease-free water in a ratio of 8:2, and set aside.

[0088] c. Add 50 μL of NEB Next Sample purification magnetic beads to the ligation product, mix by pipetting, incubate at room temperature for 5 minutes, place on a magnetic rack, and discard the supernatant after the solution becomes clear; wash with freshly prepared 80% ethanol and discard the supernatant, repeat this step once; air dry, elute with 11 μL of elution buffer, and take 10 μL of supernatant for the next reaction.

[0089] 4.5 PCR amplification and purification

[0090] 4.5.1 Add the following reagents to the purified product above to carry out the reaction.

[0091]

[0092] 4.5.2 Place it on a PCR instrument and react under the following conditions.

[0093]

[0094] 4.5.3 Purification of PCR amplification products

[0095] a. Equilibrate the purified magnetic beads at room temperature for 30 minutes, and then thoroughly mix the purified magnetic beads using a vortex mixer;

[0096] b. Prepare 80% ethanol by mixing anhydrous ethanol and nuclease-free water in a ratio of 8:2, and set aside for later use;

[0097] c. Add 22.5 μL of NEB purification magnetic beads to the ligation product, mix by pipetting, incubate at room temperature for 5 minutes, place on a magnetic rack, and discard the supernatant after the solution becomes clear; wash with freshly prepared 80% ethanol and discard the supernatant, repeat this step once; air dry, elute with 15 μL of elution buffer, and take 14 μL of supernatant for quality inspection.

[0098] 5. Hybridization elution

[0099] 5.1 Hybridization:

[0100] a. Take 10 ng of each library to be hybridized, calculate the volume of the libraries required for merging and hybridizing, and mix the 12 libraries together;

[0101] b. Add the following reagents to the library mixture to initiate the reaction;

[0102] c. Turn on the vacuum concentrator, set the V-AQ mode, and concentrate it into dry powder at room temperature;

[0103] d Immediately add 20 μL of hybridization mixture to the concentrated dry powder, mix well and let stand at room temperature for 5 minutes, then add 30 μL of hybridization enhancement solution, mix well and centrifuge;

[0104] e. Place the sample on a PCR instrument and perform the reaction under the following conditions.

[0105] Reagent composition table

[0106]

[0107] PCR reaction conditions table

[0108]

[0109] 5.2 Capture and Elution

[0110] 5.2.1 Buffer preheating:

[0111] Preheat the rapid binding solution and rapid washing solution 2 at 48°C until the precipitate dissolves, preheat the rapid washing solution 1 at 63°C until the precipitate dissolves, and equilibrate the streptavidin magnetic beads at room temperature for at least 30 minutes.

[0112] 5.2.2 Magnetic Bead Capture

[0113] a. Vortex oscillation to mix streptavidin magnetic beads that have been equilibrated to room temperature;

[0114] b. Add 70 μL of streptavidin magnetic beads to a 1.5 mL centrifuge tube;

[0115] c. Add 20 μL of binding buffer, mix by pipetting, centrifuge briefly, place on a magnetic rack, let stand for 1 min, and discard the supernatant.

[0116] d. Repeat steps b and c twice, for a total of three streptavidin magnetic bead cleaning operations;

[0117] e. Resuspend the streptavidin magnetic beads in 20 μL of binding buffer;

[0118] f. Transfer 20 μL of resuspended streptavidin magnetic beads to a hybridization reaction tube, and then transfer all the liquid to a 1.5 mL centrifuge tube.

[0119] g. Mix at room temperature for 30 minutes using a mixer, without vortexing;

[0120] After mixing, centrifuge briefly, place on a magnetic rack, let stand for 1 minute, and discard the supernatant.

[0121] 5.2.3 Washing

[0122] Add 200 μL of preheated (54℃) rapid washing buffer 1, mix well by pipetting, and incubate in a constant temperature metal bath at 54℃ for 5 min.

[0123] b. After incubation, briefly centrifuge and transfer all liquid to a new 1.5 mL centrifuge tube to remove non-specific capture fragments bound to the surface of the centrifuge tube;

[0124] c. Place on a magnetic rack and let stand for 1 minute, then discard the supernatant;

[0125] d. Add 200 μL of preheated (63℃) washing buffer 1, mix well by pipetting, and incubate in a constant temperature metal bath at 54℃ for 5 minutes.

[0126] e After incubation, centrifuge briefly, place on a magnetic rack, let stand for 1 minute, and discard the supernatant;

[0127] f Add 200 μL of preheated (54°C) washing buffer 2, mix well by pipetting, and incubate in a constant temperature metal bath at 54°C for 5 minutes.

[0128] g After incubation, centrifuge briefly, place on a magnetic rack, let stand for 1 minute, and discard the supernatant;

[0129] h Repeat steps a to g twice, for a total of three cleanings;

[0130] i. Centrifuge briefly, discard the residual supernatant with a 10μL pipette tip, immediately add 45μL of nuclease-free water, mix well by pipetting, and incubate on ice.

[0131] 5.3 PCR enrichment of capture products

[0132] 5.3.1 Take 22.5 μL of the capture product (streptavidin magnetic bead suspension) and add the following reagents to carry out the reaction.

[0133]

[0134] Place it on a PCR instrument and perform the reaction under the following conditions.

[0135]

[0136] 5.3.2 Purification of PCR Products

[0137] a. Equilibrate the DNA purification magnetic beads at room temperature for 30 minutes, then mix thoroughly using a vortex mixer;

[0138] b. Prepare 80% ethanol by mixing anhydrous ethanol and nuclease-free water in a ratio of 8:2, and set aside for later use;

[0139] c. Add 90 μL of DNA purification magnetic beads to the ligation product, mix by pipetting, incubate at room temperature for 5 minutes, place on a magnetic rack, and discard the supernatant after the solution becomes clear; wash with freshly prepared 80% ethanol and discard the supernatant, repeat this step once; air dry, elute with 32 μL of elution buffer, aspirate 30 μL of supernatant for quality control, and then sequence.

[0140] 6. Analysis of sequencing data:

[0141] The raw data from the sequencing process were first filtered using FastQC to remove low-quality sequences (phred33 score ≤ 20) as well as adapter sequences and polyA / T sequences. The filtered high-quality reads were then back-applied to the hg38 human genome using BSMAP, and reads with high back-applied quality were selected. PCR repeats were removed using Picard, and then reads back-applied to the target genomic regions (regions corresponding to DNA methylation markers) were selected using SAMTools, and the methylation levels of each DNA methylation marker were calculated.

[0142] The NGS platform methylation biomarker screening consists of two steps. The first step involves preliminary site screening using a custom large methylation panel. The samples used in this step are detailed in Table 1 below:

[0143]

[0144] Based on the results of the first step of tissue sequencing analysis, a customized capture probe panel with a size of 480K was designed.

[0145] Example 3: Screening for optimal loci in NGS-generated hepatocellular carcinoma plasma samples using a 480K panel.

[0146] The NGS test procedure is the same as that in Example 2.

[0147] Based on the 480K panel, we further screened for differential methylation sites. NGS sequencing analysis based on the 480K methylation panel was performed; sample details are shown in the table below.

[0148]

[0149] After screening the NGS sequencing results using the 480K hybridization capture panel, we identified the following gene regions as candidate biomarkers for subsequent qPCR.

[0150] The sensitivity and specificity of the candidate regions in ex vivo plasma samples of hepatocellular carcinoma are shown in the table below:

[0151]

[0152] like Figure 2 As shown, the AUC value of the determined methylation tri-labeled composition for methylation sequencing detection in ex vivo blood samples of liver cancer was 0.955.

[0153] Example 4: Validating the performance of candidate sites using a qPCR platform

[0154] To further validate the performance of the selected gene region, we used the qPCR platform to validate the selected region. The sample details of this example are shown in the table below:

[0155]

[0156] This embodiment uses methylation-specific PCR to detect the methylation level of genes in ex vivo plasma samples from liver cancer. The specific steps are as follows:

[0157] 1. Plasma separation: First, centrifuge the collected 10mL clinical blood sample at 1600×g for 10 minutes to separate the plasma. Then, centrifuge the separated plasma at 16000×g for 10 minutes to separate impurities. The purified plasma sample after impurity separation should be stored in a -80℃ refrigerator for later use.

[0158] 2. Free DNA extraction: Free DNA was extracted from the separated plasma sample using the Free DNA Extraction Kit (Catalog No.: C03-2) from Guangzhou Youze Biotechnology Co., Ltd.

[0159] 3. Cell-free DNA transformation: The extracted cell-free DNA samples were methylated to C->T using ZYMO's EZ-96 DNA Methvlation-LightningMagPrep.

[0160] 4. Negative and positive controls for quantitative PCR of methylation: The negative control was genomic DNA from a cell line transformed with the ZYMO kit (cell line: QSG-7701) at a concentration of 1 ng / μL. In the negative control, C at methylated CpG positions remained unchanged after transformation. The positive control was genomic DNA from a cell line transformed with bisulfite using the ZYMO kit (cell line: HepG2) at a concentration of 1 ng / μL. In the positive control, C at methylated CpG positions remained unchanged after transformation, while C at other positions changed to T.

[0161] 5. Reaction system for quantitative methylation PCR:

[0162]

[0163] 6. Reaction procedure for quantitative PCR of methylation:

[0164]

[0165] 7. Detection: Mix the DNA sample to be tested, negative control, and positive control according to the reaction system of methylation quantitative PCR in step 5. Place the sample into the Shanghai Hongshi SLAN-96S quantitative PCR instrument, set the instrument according to the program conditions in step 6, and collect the fluorescence values ​​of each channel (HEX / ROX / FAM / CY5).

[0166] 8. Results Recording: After the methylation quantitative PCR reaction is completed, the threshold is set at the inflection point of the S-shaped amplification curve to obtain the Ct value of the sample at the methylation site.

[0167] 9. Quality Control: Negative and positive controls are tested simultaneously for each assay. The negative control is genomic DNA from transformed cells (cell line: QSG-7701) containing the β-actin gene and a hypomethylated target gene sequence, while the positive control is cfDNA from transformed cells (cell line: HepG2) containing the β-actin gene and a hypermethylated target gene sequence, both at a concentration of 1 ng / μL. If the following conditions are met: no amplification in the negative control, a Ct value between 28 and 32 in the positive control, and a Ct value less than 38 for the internal control gene, the experiment is considered valid and the next step of sample result determination can proceed. Otherwise, the result is invalid and the assay must be repeated.

[0168] 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 used for joint determination, if the Ct value of at least one target marker is less than 35, the test result is positive; otherwise, the test result is negative.

[0169] This embodiment also provides a diagnostic reagent for primary hepatocellular carcinoma, which comprises the following nucleic acid combination:

[0170] Nucleic acid combination 1 includes primer pair 1 and probe 1. The primer pair includes upstream primer sequence 1 and downstream primer sequence 2, and probe 1 sequence 3, which are used to amplify the fragment between regions 1.

[0171] Nucleic acid combination 2 includes primer pair 2 and probe 2. The primers include upstream primer sequence 4 and downstream primer sequence 5, as well as probe 2 sequence 6, which are used to amplify the fragment between regions 2.

[0172] Nucleic acid combination 3 includes primer pair 3 and probe 3. The primer pair includes upstream primer sequence 7 and downstream primer sequence 8, and probe 3 sequence 9, which are used to amplify the fragment between regions 3.

[0173] Nucleic acid combination 4 includes primer pair 4 and probe 4. The primer pair includes upstream primer sequence 10 and downstream primer sequence 11, and probe sequence 12, which are used to amplify the fragment between regions 4.

[0174] Nucleic acid combination 5 includes primer pair 5 and probe 5. The primer pair includes upstream primer sequence 13 and downstream primer sequence 14, and probe 5 sequence 15, which are used to amplify the fragment between regions 5.

[0175] Nucleic acid combination 6 includes primer pair 6 and probe 6. The primer pair includes upstream primer sequence 16 and downstream primer sequence 17, and probe 6 sequence 18, which are used to amplify the fragment between regions 6.

[0176] Nucleic acid combination 7 includes primer pair 7 and probe 7. The primer pair includes upstream primer sequence 19 and downstream primer sequence 20, and probe 7 sequence 21, which are used to amplify the fragment between regions 7.

[0177] Nucleic acid combination 8 includes primer pair 8 and probe 8. The primer pair includes upstream primer sequence 22 and downstream primer sequence 23, and probe 8 sequence 24, which are used to amplify the fragment between regions 8.

[0178] Nucleic acid combination 9 includes primer pair 9 and probe 9. The primer pair includes upstream primer sequence 25 and downstream primer sequence 26, and probe sequence 27, which are used to amplify the fragment between regions 9.

[0179] Nucleic acid combination 10 includes primer pair 10 and probe 10. The primer pair includes upstream primer sequence 28 and downstream primer sequence 29, and probe 10 sequence 30, which are used to amplify the fragment between regions 10.

[0180] The detection performance of different nucleic acid combinations on the qPCR platform for ex vivo liver cancer blood samples is shown in the table below:

[0181]

[0182] The plasma qPCR results show that the sensitivity of using methylation markers chr5 (42992299-42992764), chr14 (53955979-53956219), and chr2 (63054088-63054326) for detecting liver cancer in ex vivo blood samples is 78.1% to 87.5%, with a specificity of approximately 90%. When all three markers are combined, the detection sensitivity can be increased to 90.6% to 93.8%, while the specificity remains around 90%.

[0183] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A three-differentiated methylation marker composition for detecting liver cancer / early stage liver cancer, characterized by, The three differential methylation marker compositions include methylation levels for simultaneously detecting the following three regions: chr5:42992299-42992764 and chr14:53955979-53956219 and chr2:63054088-63054326; The genomic locations of the three differential methylation marker compositions are all determined based on human whole genome sequence hg38 alignment; The methylation markers for detecting the region of chr5:42992299-42992764 are selected from at least one region containing CpG sites as follows: region 1: chr5:42992299-42992414 and / or region 2: chr5:42992451-42992557 and / or region 3: chr5:42992522-42992764 and / or region 4: chr5:42992565-42992764; The methylation markers for detecting the region of chr14:53955979-53956219 are selected from at least one region containing CpG sites as follows: region 5: chr14:53955979-53956053 and / or region 6: chr14:53956044-53956128 and / or region 7: chr14:53956135-53956219; The methylation markers for detecting the region of chr2:63054088-63054326 are selected from at least one region containing CpG sites as follows: region 8: chr2:63054088-63054212 and / or region 9: chr2:63054112-63054305 and / or region 10: chr2:63054194-63054326.

2. Use of the three differential methylation marker compositions for detecting liver cancer / early liver cancer according to claim 1 in the preparation of a product for detecting liver cancer / early liver cancer.

3. A tri-differential methylation kit for detecting liver cancer / early stage liver cancer, characterized by, The kit comprises the three differential methylation marker compositions for detecting liver cancer / early liver cancer according to claim 1.

4. A nucleic acid composition for detecting liver cancer / early stage liver cancer, characterized by comprising a nucleic acid encoding a polypeptide of SEQ ID NO: 1 or a fragment thereof. The nucleic acid composition comprises nucleic acid combination A and nucleic acid combination B and nucleic acid combination C; The nucleic acid combination A comprises: nucleic acid combination 1 and / or nucleic acid combination 2 and / or nucleic acid combination 3 and / or nucleic acid combination 4; The nucleic acid combination 1 for detecting the methylation level in region 1 comprises upstream and downstream primers, the nucleotide sequences of which are as shown in SEQ ID NO. 1-2, and a detection probe, the nucleotide sequence of which is as shown in 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 as shown in SEQ ID NO. 4-5, and a detection probe, the nucleotide sequence of which is as shown in 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 as shown in SEQ ID NO. 7-8, and a detection probe, the nucleotide sequence of which is as shown in SEQ ID NO. 9; The nucleic acid combination 4 for detecting the methylation level in region 4 comprises upstream and downstream primers, the nucleotide sequences of which are as shown in SEQ ID NO. 10-11, and a detection probe, the nucleotide sequence of which is as shown in SEQ ID NO.

12. The nucleic acid combination 4 for detecting the methylation level in region 4 comprises upstream and downstream primers, nucleotide sequences as SEQ ID NO. 10-11, and a detection probe, nucleotide sequence as SEQ ID NO. 12; The nucleic acid combination B comprises: nucleic acid combination 5 and / or nucleic acid combination 6 and / or nucleic acid combination 7; The nucleic acid combination 5 for detecting the methylation level in region 5 comprises upstream and downstream primers, nucleotide sequences as SEQ ID NO. 13-14, and a detection probe, nucleotide sequence as SEQ ID NO. 15; The nucleic acid combination 6 for detecting the methylation level in region 6 comprises upstream and downstream primers, nucleotide sequences as SEQ ID NO. 16-17, and a detection probe, nucleotide sequence as SEQ ID NO. 18; The nucleic acid combination 7 for detecting the methylation level in region 7 comprises upstream and downstream primers, nucleotide sequences as SEQ ID NO. 19-20, and a detection probe, nucleotide sequence as SEQ ID NO. 21; The nucleic acid combination C comprises: nucleic acid combination 8 and / or nucleic acid combination 9 and / or nucleic acid combination 10; The nucleic acid combination 8 for detecting the methylation level in region 8 comprises upstream and downstream primers, nucleotide sequences as SEQ ID NO. 22-23, and a detection probe, nucleotide sequence as SEQ ID NO. 24; The nucleic acid combination 9 for detecting the methylation level in region 9 comprises upstream and downstream primers, nucleotide sequences as SEQ ID NO. 25-26, and a detection probe, nucleotide sequence as SEQ ID NO. 27; The nucleic acid combination 10 for detecting the methylation level in region 10 comprises upstream and downstream primers, nucleotide sequences as SEQ ID NO. 28-29, and a detection probe, nucleotide sequence as SEQ ID NO.

30.

5. A nucleic acid kit for detecting liver cancer / early stage liver cancer, characterized by, The nucleic acid kit comprises the nucleic acid composition of claim 4, dNTPs, Mg 2+ , methylation-sensitive restriction enzymes, and PCR buffer.

6. The nucleic acid kit for detecting liver cancer / early stage liver cancer according to claim 5, characterized by, The liver cancer includes primary liver cancer or intrahepatic cholangiocarcinoma or mixed hepatocellular carcinoma-intrahepatic cholangiocarcinoma.

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