Methylation biomarker composition, nucleic acid composition for liver cancer detection and applications

Abstract

The present invention discloses a methylation biomarker composition, a nucleic acid composition and a kit thereof for liver cancer detection, belonging to the field of biological detection. The biomarker composition of the present invention comprises a total of six methylation markers, namely chr2:25216400-25216752, chr6:26250469-26250942, chr17:30970348-30970741, chr5:42992299-42992764, chr14:53955979-53956219, and chr2:63054088-63054326. The above marker combination and detection reagent assist in the diagnosis of liver cancer by detecting the methylation level of molecular markers in a sample. The sensitivity of the detection sample can reach more than 95%, and the specificity can reach 90%, providing a more accurate and reliable means for the early diagnosis and screening of liver cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to the application of a methylation biomarker composition, a nucleic acid composition and a kit thereof for liver cancer detection. Background Art

[0002] Primary hepatocellular carcinoma (HCC) is one of the common malignant tumors worldwide and also the most common cause of death in patients with chronic liver disease. The onset of liver cancer mostly follows the "three-step" pattern of hepatitis B - liver cirrhosis - liver cancer, and about 85% of HCC is caused by HBV infection.

[0003] With the improvement of the diagnosis and treatment technology level, great progress has been made in the prevention and treatment of HCC. However, due to the insidious onset and rapid progression of HCC, most cases are diagnosed at the middle and advanced stages. Therefore, early screening and diagnosis of HCC have become the key. AFP is one of the serum markers for diagnosing liver cancer, but about 30% of liver cancer patients always have negative serum AFP, and AFP also increases in some high-risk populations of liver cancer such as chronic hepatitis and liver cirrhosis. AFP has a relatively high false negative rate in the early detection of liver cancer and is not very ideal as an early screening index for liver cancer.

[0004] The detection of peripheral blood cfDNA methylation is another non-invasive early diagnosis method for tumors in recent years. Compared with the blood AFP level, free DNA methylation markers have higher sensitivity and specificity for liver cancer detection. Although some free DNA methylation markers for liver cancer have been preliminarily reported in some studies, most of these markers are screened from 450K methylation chip data, covering less than 2% of the human genome methylation sites.

[0005] Therefore, using a more comprehensive biomarker screening strategy and method is the key to developing more accurate liver cancer methylation markers. Summary of the Invention

[0006] The purpose of the present invention is to provide a methylation biomarker composition for liver cancer detection. The DNA methylation markers and compositions used in the kit are screened from genomic regions with a 10-fold coverage of the 450K methylation chip, and the detection performance of the markers and compositions screened in the sample dataset used in the present invention is optimal.

[0007] The present invention is achieved through the following technical solutions:

[0008] A methylation biomarker composition for liver cancer detection, wherein the methylation biomarker composition is a combination of chr2:25216400-25216752, chr6:26250469-26250942, chr17:30970348-30970741, chr5:42992299-42992764, chr14:53955979-53956219, and chr2:63054088-63054326;

[0009] The methylation biomarker is derived from the hg38 human genome;

[0010] The methylation level is the methylation level of CpG sites on the molecular biomarker.

[0011] A nucleic acid composition for liver cancer detection, comprising a first nucleic acid combination for detecting the methylation of chr2:25216400-25216752, a second nucleic acid combination for detecting the methylation of chr6:26250469-26250942, a third nucleic acid combination for detecting the methylation of chr17:30970348-30970741, a fourth nucleic acid combination for detecting the methylation of chr5:42992299-42992764, a fifth nucleic acid combination for detecting the methylation of chr14:53955979-53956219, and a sixth nucleic acid combination for detecting the methylation of chr2:63054088-63054326.

[0012] Preferably, the first nucleic acid combination includes primer pair 1, and the base sequence of primer pair 1 has at least 90% identity with the base sequences shown in SEQ ID NO.1-2;

[0013] The second nucleic acid combination includes primer pair 2, and the base sequence of primer pair 2 has at least 90% identity with the base sequences shown in SEQ ID NO.3-4;

[0014] The third nucleic acid combination includes primer pair 3, and the base sequence of primer pair 3 has at least 90% identity with the base sequences shown in SEQ ID NO.5-6;

[0015] The fourth nucleic acid combination includes primer pair 4, and the base sequence of primer pair 4 has at least 90% identity with the base sequences shown in SEQ ID NO.7-8;

[0016] The fifth nucleic acid combination includes primer pair 5, and the base sequence of primer pair 5 has at least 90% identity with the base sequences shown in SEQ ID NO.9-10;

[0017] The sixth nucleic acid combination includes primer pair 6, and the base sequence of primer pair 6 has at least 90% identity with the base sequences shown in SEQ ID NO.11-12.

[0018] Preferably, the first nucleic acid combination further includes probe 1, and the base sequence of probe 1 has at least 90% identity with the base sequence shown in SEQ ID NO.13.

[0019] The second nucleic acid combination further includes probe 2, and the base sequence of probe 2 has at least 90% identity with the base sequence shown in SEQ ID NO.14.

[0020] The third nucleic acid combination further includes probe 3, and the base sequence of probe 3 has at least 90% identity with the base sequence shown in SEQ ID NO.15.

[0021] The fourth nucleic acid combination further includes probe 4, and the base sequence of probe 4 has at least 90% identity with the base sequence shown in SEQ ID NO.16.

[0022] The fifth nucleic acid combination further includes probe 5, and the base sequence of probe 5 has at least 90% identity with the base sequence shown in SEQ ID NO.17.

[0023] The sixth nucleic acid combination further includes probe 6, and the base sequence of probe 6 has at least 90% identity with the base sequence shown in SEQ ID NO.18.

[0024] A reagent product for detecting liver cancer, wherein the reagent product contains the methylation biomarker composition and the nucleic acid composition described above.

[0025] Preferably, the reagent product is any one of a reagent, a kit, a chip, and a sequencing library.

[0026] A kit for detecting liver cancer, when the reagent product is a kit, it further includes a positive control, a negative control, detection primers for an internal reference gene, a detection probe for an internal reference gene, a DNA polymerase, and a buffer.

[0027] Preferably, the internal reference gene is the ACTB gene.

[0028] Preferably, the detection sample of the kit includes an ex vivo blood sample; the ex vivo blood sample is selected from an ex vivo plasma sample, an ex vivo serum sample, an ex vivo whole blood sample, or an ex vivo blood cell sample.

[0029] A fluorescent reporter group is labeled at the 5' end of the above-mentioned probe, and a fluorescent quenching group is labeled at the 3' end of the above-mentioned probe. Fluorescent labeling is used to better achieve signal amplification and high-sensitivity detection.

[0030] In one embodiment, the above-mentioned probe is a Taqman probe.

[0031] The above-mentioned fluorescent reporter group is HEX, FAM, TET, CF532, JOE, TAMRA, ROX, CY3, CY5, Texas Red, NED, Alexa Flour or VIC, and the quenching group is MGB, TAMRA, BHQ1, BHQ2, BHQ3 or QSY.

[0032] In a preferred embodiment of the application of the present invention, the above-mentioned methylation level is detected by at least one of the following methods: methylation-specific PCR method, sequencing method, methylation-specific high-performance liquid chromatography method, digital PCR method, methylation-specific high-resolution melting curve method, methylation-specific microarray method, methylation-sensitive restriction enzyme method and flapendonucleas method.

[0033] The above-mentioned sequencing method is selected from bisulfite sequencing method, whole-genome methylation sequencing, targeted methylation sequencing method or pyrosequencing method.

[0034] Compared with the prior art, the present invention has at least the following technical effects:

[0035] (1) The present invention provides a combination of liver cancer molecular markers and a detection kit. The molecular marker combination is a composition including six biomarkers, namely chr2:25216400-25216752, chr6:26250469-26250942, chr17:30970348-30970741, chr5:42992299-42992764, chr14:53955979-53956219, and chr2:63054088-63054326, which is used to detect the signal of abnormal methylation increase in liver cancer and has ideal detection specificity and sensitivity.

[0036] (2) The present invention combines multiple molecular markers related to the detection of early liver cancer to predict early liver cancer, and can further significantly improve the detection rate of early liver cancer on the basis of a single methylation marker, avoiding the problems of false detection and missed detection.

[0037] (3) The DNA methylation markers and compositions used in the kit are screened from genomic regions with a coverage 10 times that of the 450K methylation chip, and the detection performance of the screened markers and compositions is optimal in the sample dataset used in the present invention.

[0038] (4) During the biomarker screening process, the methylation sequencing adopted is based on an enzyme conversion method for the conversion of cytosine to uracil. Compared with the traditional bisulfite conversion, it can avoid DNA damage, thus solving the problem of low content of cell-free DNA in blood.

[0039] (5) The molecular marker combination and its kit provided by the present invention are simple to operate and low in detection cost compared with large-scale NGS sequencing, and can quickly and accurately perform reasonable auxiliary discrimination on liver cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the R & D flow chart for the discovery and screening of methylation markers for detecting liver cancer in Example 1;

[0041] Figure 2 is the ROC graph of the detection of the methylation marker composition in the ex vivo blood samples of liver cancer in Example 3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0042] The following will describe the embodiments of the present invention in detail in conjunction with the 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 regarded as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not specified in the manufacturer are all conventional products that can be obtained through commercial purchase.

[0043] Example 1: Screening Process of Methylation Markers for Liver Cancer Detection

[0044] This example discloses a methylation marker composition for detecting early liver cancer and its screening process. The specific screening process is as Figure 1 shown.

[0045] In order to discover and screen methylation markers with excellent detection performance, this example first adopted a customized hybridization capture large panel for targeted methylation sequencing. This hybridization capture panel covers 3.98 million CpG methylation sites in the human genome, which covers most of the CpG island regions and genes related to cancer occurrence, and the coverage range is 10 times that of the traditional DNA methylation chip. By performing targeted sequencing on 82 clinical frozen tissue samples and 115 ex vivo blood samples and comparing the changes in methylation patterns between the cancer group and the non-cancer group, this example initially determined 30,008 methylation sites that can significantly distinguish cancer samples and non-cancer samples in both tissue samples and blood samples as potential cancer markers.

[0046] Furthermore, a hybridization capture panel was designed based on the 30,008 methylated sites preliminarily screened above for the second round of targeted methylation sequencing. This round of sequencing adopted a deep sequencing strategy with an average sequencing depth reaching 500X, significantly improving the accuracy of methylation level calculation and facilitating the precise screening of biomarkers in the second round. Targeted methylation sequencing was performed on the ex vivo blood samples of 218 clinical liver cancer patients and the ex vivo blood samples of 196 non-cancer groups, and 12 DNA methylation markers with the best liver cancer detection performance were precisely screened using univariate statistical analysis and machine learning methods.

[0047] Finally, primer and probe nucleic acid combinations for the qPCR detection platform were designed for the genomic regions of the 12 selected methylation markers, and the performance verification of liver cancer detection was carried out on the qPCR platform using the ex vivo blood samples of 32 clinical liver cancer patients and the ex vivo blood samples of 47 non-cancer groups. The composition of the DNA methylation markers with the best liver cancer detection performance was determined through univariate performance analysis and machine learning feature selection methods, that is, the composition of six biomarkers: chr2:25216400-25216752, chr6:26250469-26250942, chr17:30970348-30970741, chr5:42992299-42992764, chr14:53955979-53956219, chr2:63054088-63054326. The primer-probe combinations for detecting the methylation level of this marker composition include:

[0048] The first nucleic acid combination for detecting the methylation of chr2:25216400-25216752, the second nucleic acid combination for detecting the methylation of chr6:26250469-26250942, the third nucleic acid combination for detecting the methylation of chr17:30970348-30970741, the fourth nucleic acid combination for detecting the methylation of chr5:42992299-42992764, the fifth nucleic acid combination for detecting the methylation of chr14:53955979-53956219, and the sixth nucleic acid combination for detecting the methylation of chr2:63054088-63054326;

[0049] The first nucleic acid combination includes primer pair 1 (including the upstream primer and the downstream primer) and probe 1,

[0050] The base sequence of the upstream primer of primer pair 1: 5’-AGACGGAGTTTTAATTTTGTTGT-3’ (SEQ ID NO.1)

[0051] The base sequence of the downstream primer of primer pair 1: 5’-TCGCTTAAATCTAAAAAACGA-3’ (SEQ ID NO.2)

[0052] The base sequence of probe 1: 5’-gaaatcgcgccattacaccg-3’ (SEQ ID NO.13);

[0053] The second nucleic acid combination includes primer pair 2 (including an upstream primer and a downstream primer) and probe 2,

[0054] The base sequence of the upstream primer of primer pair 2: 5’-CTTCCAATTTCGCGCC-3’ (SEQ ID NO.3)

[0055] The base sequence of the downstream primer of primer pair 2: 5’-ACGCGGTATTTTTGAATCG-3’ (SEQ ID NO.4)

[0056] The base sequence of probe 2: 5’-CAAACGAAAATAAACGTTCTACAATTCCG-3’ (SEQ ID NO.14);

[0057] The third nucleic acid combination includes primer pair 3 (including an upstream primer and a downstream primer) and probe 3,

[0058] The base sequence of the upstream primer of primer pair 3: 5’-TTTGTTGGGAAACGCGTTTA-3’ (SEQ ID NO.5)

[0059] The base sequence of the downstream primer of primer pair 3: 5’-TTTTAACTCGCAAATATAAT-3’ (SEQ ID NO.6)

[0060] The base sequence of probe 3: 5’-AAGGCGGTAAGGATATAGCG-3’ (SEQ ID NO.15);

[0061] The fourth nucleic acid combination includes primer pair 4 (including an upstream primer and a downstream primer) and probe 4,

[0062] The base sequence of the upstream primer of primer pair 4: 5’-AGTTCGTTGTTCGATGTTTTTATT-3’ (SEQ ID NO.7)

[0063] The base sequence of the downstream primer of primer pair 4:

[0064] 5’-CTACGACGACGCTAAAAAAAAACGA-3’ (SEQ ID NO.8)

[0065] Probe 4 base sequence: 5’-TCGGGGCGATAGTATTTAGGTCG (SEQ ID NO.16);

[0066] The fifth nucleic acid combination includes primer pair 5 (including an upstream primer and a downstream primer) and probe 5,

[0067] Upstream primer base sequence of primer pair 5: 5’-GTTGCGATTTTCGGACGGA-3’ (SEQ ID NO.9)

[0068] Downstream primer base sequence of primer pair 5: 5’-CGTCGCTCTACCCCCTCA-3’ (SEQ ID NO.10)

[0069] Probe 5 base sequence: 5’-GCGGGAGGAGGAAGGAGA-3’ (SEQ ID NO.17);

[0070] The sixth nucleic acid combination includes primer pair 6 (including an upstream primer and a downstream primer) and probe 6,

[0071] Upstream primer base sequence of primer pair 6: 5’-TTTAGGTGCGTATTTTTCGG-3’ (SEQ ID NO.11)

[0072] Downstream primer base sequence of primer pair 6: 5’-CGACTACCCAATCTATAAACC-3’ (SEQ ID NO.12)

[0073] Probe 6 base sequence: 5’-GAGGTTGTTCGGGGAAGGTTTAG-3’ (SEQ ID NO.18).

[0074] Example 2: Detection performance of the screened markers in liver cancer tissue samples

[0075] Collect 33 clinical samples of liver cancer tissue and 49 non-cancer control tissue samples, and perform genomic extraction, enzyme conversion-based methylation conversion, and targeted methylation sequencing on the tissue samples. The specific experimental steps are as follows:

[0076] 1. Extraction of DNA samples: When the sample is a frozen fresh tissue sample, use the 《DNeasy® Blood&Tissue Kit #69506》 from QIAGEN to extract the genome. The specific operation refers to the kit instruction manual. The genomic DNA is fragmented using a Covaris M220 ultrasonic disruptor. When extracting cfDNA from plasma samples, use the 《Cell-free DNA Extraction Kit (filtration method) #C02-1》 from Guangzhou Youze Biotechnology Co., Ltd. The specific operation refers to the kit instruction manual.

[0077] 2. Screen the extracted DNA samples using SPRIselect beads according to the recommended steps in the instruction manual to filter out DNA molecules larger than 200 bp.

[0078] 3. Quality control product preparation: Add 10 µL of 0.1 ng / µL CpG-methylated pUC19 and 10 µL of 2 ng / µL non-methylated λDNA to each sample.

[0079] 4. Library construction for methylation sequencing: Use the NEBNext Enzymatic Methyl-Seq kit to construct the library.

[0080] 4.1 End repair and addition of "A" at the 3' end

[0081] 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.

[0082]

[0083] 4.1.2 Place it in a PCR instrument and perform the reaction according to the following program

[0084]

[0085] 4.2 Adapter ligation

[0086] 4.2.1 Add the following reagents to the end repair product for reaction

[0087]

[0088] 4.2.2 Place it in a PCR instrument and set the PCR reaction program according to the following conditions: 60°C, 60 min, heat lid closed.

[0089] 4.2.3 Purification of adapter ligation product

[0090] A. Equilibrate the purification beads at room temperature for 30 min and mix the purification beads thoroughly on a vortex mixer.

[0091] B. Prepare 80% ethanol by mixing absolute ethanol and nuclease-free water in a ratio of 8:2 and set aside.

[0092] C. Add 55 μL of purification beads to the ligation product respectively, pipette to mix well, incubate at room temperature for 5 minutes, place on a magnetic stand, and discard the supernatant after the solution becomes clear; wash with the freshly prepared 80% ethanol and discard the supernatant, repeat this step once; air dry, elute with 15 μL of elution buffer, and pipette 14 μL of the supernatant for the next reaction.

[0093] 4.3 Methylation: Oxidation reaction

[0094] 4.3.1 Preparation of buffer:

[0095] a. Add 400 μL of E1 reaction buffer supplement to 100 μL of E1 reaction buffer and mix well by shaking. Mark the preparation date.

[0096] 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 freshly prepared. Do not store.

[0097] c. Dilute the stop buffer with nuclease-free water at a ratio of 1:10.

[0098] 4.3.2 Add the following reagents to 14 μL of the purified ligation product and mix well. Then add 10 μL of the diluted Fe(II) to the purified ligation product with oxidase added and mix well, followed by centrifugation.

[0099]

[0100] 4.3.3 Place it on a PCR instrument and react under the following conditions: 37 °C, 1 h, hot lid temperature ≥ 45 °C.

[0101] 4.3.4 After the reaction is completed, add 1 μL of the diluted reaction stop solution to the product and place it on a PCR instrument. React under the following conditions: 37 °C, 30 min, hot lid temperature ≥ 45 °C.

[0102] 4.3.5 Purification of the oxidation reaction product

[0103] A. Equilibrate the purification magnetic beads at room temperature for 30 min and mix well on a vortex mixer.

[0104] B. Prepare 80% ethanol by mixing absolute ethanol and nuclease-free water at a ratio of 8:2 for later use.

[0105] C. Add 45 μL of NEB Next Sample purification magnetic beads to the ligation product respectively, pipette and mix well, incubate at room temperature for 5 minutes, place on a magnetic stand, 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 pipette 8 μL of the supernatant for the next reaction.

[0106] 4.4 Methylation: Cytosine deamination

[0107] 4.4.1 Denaturation:

[0108] a. Preheat the PCR instrument to 85 °C and turn on the hot lid.

[0109] Add 2 μL of formamide to 8 μL of the purified oxidation reaction product, vortex to mix well, and centrifuge briefly.

[0110] 4.4.2 Add the following reagents to the denatured product for reaction

[0111]

[0112] 4.4.3 Place it on a PCR instrument and react under the following conditions: 37°C, 3 h, hot lid temperature ≥ 45°C.

[0113] 4.4.4 Purification of the cytosine deamination reaction product

[0114] a Equilibrate the purification magnetic beads at room temperature for 30 min and mix the purification magnetic beads thoroughly on a vortex mixer;

[0115] b Prepare 80% ethanol according to the ratio of absolute ethanol: nuclease-free water = 8:2 and set aside;

[0116] c Add 50 μL of NEB Next Sample purification magnetic beads to the ligation product respectively, pipette to mix well, incubate at room temperature for 5 minutes, place on a magnetic stand, and discard the supernatant after the solution becomes clear; wash with the freshly prepared 80% ethanol and discard the supernatant, repeat this step once; air dry, elute with 11 μL of elution buffer, and aspirate 10 μL of the supernatant for the next reaction.

[0117] 4.5 PCR amplification and purification

[0118] 4.5.1 Add the following reagents to the above purified product for reaction

[0119]

[0120] 4.5.2 Place it on a PCR instrument and react under the following conditions

[0121]

[0122] 4.5.3 Purification of the PCR amplification product

[0123] a Equilibrate the purification magnetic beads at room temperature for 30 min and mix the purification magnetic beads thoroughly on a vortex mixer;

[0124] b Prepare 80% ethanol according to the ratio of absolute ethanol: nuclease-free water = 8:2 and set aside;

[0125] c Add 22.5 μL of NEB purification magnetic beads to the ligation product respectively, pipette and mix well, incubate at room temperature for 5 minutes, place on a magnetic stand, 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, aspirate 14 μL of the supernatant for quality inspection.

[0126] 5. Hybridization and Elution

[0127] 5.1 Hybridization:

[0128] a Take 10 ng from each library to be hybridized, calculate the volume of the libraries required for combined hybridization, and mix 12 libraries together;

[0129] b Add the following reagents to the library mixture for reaction;

[0130] c Turn on the vacuum concentrator, set the V-AQ mode, and concentrate into dry powder at room temperature;

[0131] d Immediately take 20 μL of the hybridization mixture and add it to the concentrated dry powder, mix well and let stand at room temperature for 5 minutes, then add 30 μL of hybridization enhancer, mix well and centrifuge;

[0132] e Place it on a PCR instrument and react under the following conditions.

[0133] Reagent Composition Table

[0134]

[0135] PCR Reaction Condition Table

[0136]

[0137] 5.2 Capture and Elution

[0138] 5.2.1 Buffer Preheating:

[0139] Preheat the rapid binding buffer and rapid wash buffer 2 to dissolve the precipitate at 48 °C, and preheat the rapid wash buffer 1 to dissolve the precipitate at 63 °C. Equilibrate the streptavidin magnetic beads at room temperature for at least 30 min.

[0140] 5.2.2 Magnetic Bead Capture

[0141] a Vortex and mix the streptavidin magnetic beads that have been equilibrated to room temperature;

[0142] b Take 70 μL of streptavidin magnetic beads and add them to a 1.5 mL centrifuge tube;

[0143] c Add 20 μL of binding buffer, pipette and mix well, centrifuge briefly and then place on a magnetic stand, let stand for 1 min, and discard the supernatant;

[0144] d Repeat steps b and c twice for a total of three washes with streptavidin magnetic beads.

[0145] e Resuspend the streptavidin magnetic beads by adding 20 μL of binding buffer.

[0146] f Transfer 20 μL of the resuspended streptavidin magnetic beads to the hybridization reaction tube, and then transfer all the liquid to a 1.5 mL centrifuge tube.

[0147] g Mix on a mixer for 30 minutes at room temperature without vortexing.

[0148] h After mixing, centrifuge briefly, place on a magnetic stand, let stand for 1 minute, and discard the supernatant.

[0149] 5.2.3 Elution

[0150] a Add 200 μL of pre-warmed (54 °C) Quick Wash Buffer 1, pipette to mix, and incubate in a thermostatic metal bath at 54 °C for 5 min.

[0151] b After incubation, centrifuge briefly, transfer all the liquid to a new 1.5 mL centrifuge tube to remove non-specific capture fragments bound to the surface of the centrifuge tube.

[0152] c Place on a magnetic stand, let stand for 1 minute, and discard the supernatant.

[0153] d Add 200 μL of pre-warmed (63 °C) Wash Buffer 1, pipette to mix, and incubate in a thermostatic metal bath at 54 °C for 5 minutes.

[0154] e After incubation, centrifuge briefly, place on a magnetic stand, let stand for 1 min, and discard the supernatant.

[0155] f Add 200 μL of pre-warmed (54 °C) Wash Buffer 2, pipette to mix, and incubate in a thermostatic metal bath at 54 °C for 5 minutes.

[0156] g After incubation, centrifuge briefly, place on a magnetic stand, let stand for 1 minute, and discard the supernatant.

[0157] h Repeat steps a to g twice for a total of three washes.

[0158] i Centrifuge briefly, use a 10 μL pipette tip to discard the remaining supernatant, immediately add 45 μL of nuclease-free water, pipette to mix, and incubate on ice.

[0159] 5.3 PCR Enrichment of the Captured Product

[0160] 5.3.1 Add the following reagents to 22.5 μL of the captured product (streptavidin magnetic bead suspension) for the reaction.

[0161]

[0162] Place it on a PCR instrument and perform the reaction under the following conditions

[0163]

[0164] 5.3.2 Purification of PCR products

[0165] a Equilibrate the DNA purification magnetic beads at room temperature for 30 min and mix well on a vortex mixer;

[0166] b Prepare 80% ethanol according to the ratio of absolute ethanol: nuclease-free water = 8:2 and set aside;

[0167] c Add 90 μL of DNA purification magnetic beads to the ligation product respectively, pipette and mix well, incubate at room temperature for 5 minutes, place on a magnetic stand, 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 the supernatant for quality control, and sequence on the machine.

[0168] 6. Analysis of sequencing data:

[0169] The raw data downloaded from the machine is first used in fastQC to filter out low-quality sequences (phred33 score ≤ 20), adapter sequences and polyA / T sequences in the reads. The filtered high-quality reads are mapped to the hg38 human genome using BSMAP, and reads with higher mapping quality are selected. After removing PCR duplicates using picard, reads mapped to the target genomic region (the region corresponding to the DNA methylation markers) are selected by samtools and the methylation levels of each DNA methylation marker are calculated.

[0170] The detection performance of the identified methylation markers and the composition in differentiating liver cancer tissues and non-cancerous tissues is as follows in the table:

[0171]

[0172] Detection performance of the screened markers in liver cancer tissue samples in Example 3

[0173] Collect 414 clinical blood samples, including blood samples from 218 patients with primary liver cancer, blood samples from 173 patients with benign liver diseases, and blood samples from 23 healthy individuals. Extract cell-free DNA, perform methylation conversion and targeted methylation sequencing on the blood samples according to the method described in Example 2, and calculate the methylation levels of each methylation marker.

[0174] Statistical results of blood samples:

[0175]

[0176] Perform univariate statistical analysis on a single methylation marker and calculate the detection performance of in vitro blood samples that distinguish liver cancer patients from the control group. Combine the identified markers at chr2:25216400-25216752, chr6:26250469-26250942, chr17:30970348-30970741, chr5:42992299-42992764, chr14:53955979-53956219, chr2:63054088-63054326 for liver cancer prediction and statistically analyze the detection performance of the six-marker combination.

[0177] The liver cancer detection specificity and sensitivity of the identified methylation markers and the combination are as follows in the table:

[0178]

[0179] As Figure 2 shown, the AUC value of the identified methylation marker combination for methylation sequencing detection of in vitro blood samples of liver cancer is 0.976.

[0180] Example 4: Performance of the identified methylation markers in the qPCR detection dataset of in vitro blood samples of liver cancer

[0181] Design primer-probe combinations required for the methylation qPCR detection platform for the identified biomarkers at chr2:25216400-25216752, chr6:26250469-26250942, chr17:30970348-30970741, chr5:42992299-42992764, chr14:53955979-53956219, chr2:63054088-63054326. The design results are as follows:

[0182]

[0183] Collect 79 clinical blood samples, including blood samples from 32 patients with primary liver cancer, 32 patients with benign liver diseases, and 15 healthy individuals.

[0184]

[0185] Perform methylation quantitative qPCR detection on the above 79 collected blood samples and record the Ct values of the qPCR detection. The specific experimental steps are as follows:

[0186] 1. Plasma separation: First, centrifuge the collected 10 mL clinical blood sample at 1600×g for 10 minutes to separate plasma. Then, centrifuge the separated plasma at 16000×g for 10 minutes to separate impurities. The plasma sample after separating and purifying impurities should be stored in a -80°C refrigerator for future use.

[0187] 2. Extraction of cell-free DNA: Use the "Cell-free DNA Extraction Kit" (product number: C03-2) from Guangzhou Youze Biotechnology Co., Ltd. to extract cell-free DNA from the separated plasma sample.

[0188] 3. Conversion of cell-free DNA: Use the "EZ-96 DNA Methvlation-LightningMagPrep" from ZYMO to perform methylation C->T conversion on the extracted cell-free DNA sample.

[0189] 4. Negative and positive controls for methylation quantitative PCR: The negative control is the genomic DNA of the cell line (cell line: QSG-7701) after being converted by the ZYMO kit, with a concentration of 1 ng / μL. After conversion, all C in the negative control becomes T. The positive control is the genomic DNA of the cell line (cell line: HepG2) after being bisulfite-converted by the ZYMO kit, with a concentration of 1 ng / μL. After conversion of the positive control, C at the methylated CpG positions does not change, while C at other positions becomes T.

[0190] 5. Reaction system for methylation quantitative PCR:

[0191]

[0192] 6. Reaction program for methylation quantitative PCR:

[0193]

[0194] 7. Detection by machine: Mix the DNA of the sample to be detected, the negative reference, and the positive reference according to the reaction system of methylation quantitative PCR in step 5. Put the sample into the Shanghai Hongshi SLAN-96S quantitative PCR instrument, and set the quantitative PCR instrument according to the program conditions in step 6, and then perform detection to collect the fluorescence values of each channel of HEX / ROX / FAM / CY5.

[0195] 8. Result recording: After the methylation quantitative PCR reaction is completed, set the threshold at the inflection point of the S-shaped amplification curve to obtain the Ct value of the sample to be detected at the methylation site.

[0196] 9. Quality control: During each detection, the negative reference and positive reference are detected synchronously. The negative control is the genomic DNA of transformed cells containing the β-actin gene and the hypomethylated target gene sequence (cell line: QSG-7701), and the positive control is the cfDNA of transformed cells containing the β-actin gene and the hypermethylated target gene sequence (cell line: HepG2), with a concentration of 1 ng / μL for both. When the following conditions are met: no amplification in the negative reference, the Ct value of the positive reference is between 28 and 32, and the Ct value of the internal reference gene is less than 38, this experiment is valid and the next step of sample result determination can be carried out. Otherwise, the result is invalid and the detection must be repeated.

[0197] 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 the Ct value of at least one target marker is less than 35, the test result is positive; otherwise, the test result is negative.

[0198] The CT values obtained by qPCR detection using the designed primer-probe combination in ex vivo blood samples are used to distinguish liver cancer and non-cancer samples. The detection performance is shown in the following table.

[0199]

[0200] It can be seen from the blood qPCR detection results that when using the methylation markers at chr2:25216400 - 25216752, chr6:26250469 - 26250942, chr17:30970348 - 30970741, chr5:42992299 - 42992764, chr14:53955979 - 53956219, and chr2:63054088 - 63054326 alone to detect ex vivo blood samples of liver cancer, the detection sensitivity ranges from 81.3% to 87.5%, and the specificity is about 90%. When combining the six methylation markers, the detection sensitivity can be increased to 96.9%, and the specificity remains about 90%.

[0201] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A methylation biomarker composition for liver cancer detection, characterized in that: The methylation biomarker composition is a combination of chr2:25216400-25216752, chr6:26250469-26250942, chr17:30970348-30970741, chr5:42992299-42992764, chr14:53955979-53956219 and chr2:63054088-63054326; The methylation biomarkers are derived from the hg38 human genome; The methylation level is the methylation level of the CpG site on the molecular marker.

2. A nucleic acid composition for liver cancer detection, characterized in that: It includes a first nucleic acid combination for detecting methylation of the chr2:25216400-25216752 CpG site as described in claim 1, a second nucleic acid combination for detecting methylation of the chr6:26250469-26250942 CpG site, a third nucleic acid combination for detecting methylation of the chr17:30970348-30970741 CpG site, a fourth nucleic acid combination for detecting methylation of the chr5:42992299-42992764 CpG site, a fifth nucleic acid combination for detecting methylation of the chr14:53955979-53956219 CpG site, and a sixth nucleic acid combination for detecting methylation of the chr2:63054088-63054326 CpG site.

3. A nucleic acid composition for liver cancer detection according to claim 2, characterized in that: The first nucleic acid combination includes primer pair 1, and the primer pair 1 is an upstream primer sequence and a downstream primer sequence as shown in SEQ ID NO.1-2; The second nucleic acid combination includes primer pair 2, and the primer pair 2 is an upstream primer sequence and a downstream primer sequence as shown in SEQ ID NO.3-4; The third nucleic acid combination includes primer pair 3, and the primer pair 3 is an upstream primer sequence and a downstream primer sequence as shown in SEQ ID NO.5-6; The fourth nucleic acid combination includes primer pair 4, and the primer pair 4 is an upstream primer sequence and a downstream primer sequence as shown in SEQ ID NO.7-8; The fifth nucleic acid combination includes primer pair 5, and the primer pair 5 is an upstream primer sequence and a downstream primer sequence as shown in SEQ ID NO.9-10; The sixth nucleic acid combination includes primer pair 6, and the primer pair 6 is an upstream primer sequence and a downstream primer sequence as shown in SEQ ID NO.11-12.

4. A nucleic acid composition for liver cancer detection according to claim 3, characterized in that: The first nucleic acid combination further includes probe 1, wherein the probe 1 is a probe sequence as shown in SEQ ID NO.13; The second nucleic acid combination further includes probe 2, wherein the probe 2 is a probe sequence as shown in SEQ ID NO.14; The third nucleic acid combination further includes probe 3, wherein the probe 3 is a probe sequence as shown in SEQ ID NO.15; The fourth nucleic acid combination further includes probe 4, wherein the probe 4 is a probe sequence as shown in SEQ ID NO.16; The fifth nucleic acid combination further includes probe 5, wherein the probe 5 is a probe sequence as shown in SEQ ID NO.17; The sixth nucleic acid combination further includes probe 6, and the probe 6 is a probe sequence as shown in SEQ ID NO.

18.

5. A reagent product for liver cancer detection, characterized in that: The reagent product comprises the methylation biomarker composition according to claim 1 and the nucleic acid composition according to any one of claims 2-4.

6. A reagent product for liver cancer detection according to claim 5, characterized in that: The reagent product is any one of a reagent, a kit, a chip and a sequencing library.

7. A reagent product for liver cancer detection according to claim 6, characterized in that: The kit also includes a positive control, a negative control, a detection primer of an internal reference gene, a detection probe of an internal reference gene, a DNA polymerase and a buffer.

8. A reagent product for liver cancer detection according to claim 7, characterized in that: The internal reference gene is the ACTB gene.

9. A reagent product for liver cancer detection according to claim 7, characterized in that: The test sample includes an ex vivo blood sample; the ex vivo blood sample is selected from an ex vivo plasma sample, an ex vivo serum sample, an ex vivo whole blood sample or an ex vivo blood cell sample.

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