Molecular marker methylation level detection reagent and its application in detection products

Abstract

The present invention discloses an application of a molecular marker methylation level detection reagent in the preparation of a primary hepatocellular carcinoma detection reagent product, and belongs to the field of gene detection technology. The scheme relates to molecular liquid biopsy technology. The technology discloses that the methylation level of a CpG site in the chr2:25216400-25216900 region is related to the occurrence of primary hepatocellular carcinoma (HCC). By detecting the methylation level of the region, it can be determined whether the target sample is hepatocellular carcinoma. The above region can be used as a molecular marker for the diagnosis of primary hepatocellular carcinoma. The technology provides a good molecular marker for the early detection of primary hepatocellular carcinoma (HCC).

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and in particular relates to the application of a molecular marker methylation level detection reagent in the preparation of a primary hepatocellular carcinoma detection reagent product. Background Art

[0002] 'Early screening, early diagnosis and early treatment' of liver cancer is considered to be one of the important means to reduce the impact of liver cancer on patients and improve their survival rate, especially for small liver cancers less than 5 cm. The 5-year survival rate of patients treated early can reach more than 80%.

[0003] However, the sensitivity of the currently commonly used liver cancer screening methods, AFP and ultrasound examinations, is not ideal, resulting in serious missed detection problems. Therefore, developing a more accurate and convenient liver cancer screening method is of great significance for the popularization and promotion of early liver cancer screening.

[0004] Abnormal DNA methylation signals are one of the characteristics of cancer. In recent years, studies have shown that the abnormal methylation signals carried by free DNA in the blood have good stability and can be used as molecular markers to detect the occurrence of cancer. Compared with blood AFP levels, free DNA methylation markers have higher sensitivity and specificity for liver cancer detection. Although some studies have preliminarily reported some free DNA methylation markers for liver cancer, most of these markers were screened from 450K methylation chip data, covering less than 2% of the methylation sites in the human genome.

[0005] Therefore, using more comprehensive marker screening strategies and methods is the key to developing more accurate HCC methylation markers. Summary of the invention

[0006] The object of the present invention is to provide a molecular marker methylation level detection reagent for use in the preparation of a primary hepatocellular carcinoma detection product, which uses the hg38 genome as a reference genome, and by detecting the methylation rate of a specific region of the genome, it can be determined whether the sample is primary liver cancer. The use of this detection product can distinguish liver cancer samples from normal healthy human samples, and can also distinguish liver cancer from benign liver diseases. At the same time, the above-mentioned methylation sites can be highly sensitive for screening early liver cancer, and are methylation markers with good sensitivity and specificity for early screening of liver cancer, which can be convenient, fast, and low-cost for early screening of liver cancer samples. At the same time, the early diagnosis ratio of HCC high-risk populations can be increased, the early survival rate of HCC patients can be increased, and the morbidity and mortality rates can be reduced.

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

[0008] Application of a molecular marker methylation level detection reagent in the preparation of a primary hepatocellular carcinoma detection product, wherein the molecular marker is a nucleic acid molecule containing at least one CpG site in the chr2:25216400-25216900 genomic region sequence;

[0009] The genomic region is from the hg38 human reference genome;

[0010] The molecular marker is located in the following regions containing at least one CpG site: region 1, region 2, region 3, region 4, and region 5;

[0011] The region 1 is chr2: 25216400- 25216504; the region 2 is chr2: 25216454- 25216521; the region 3 is chr2: 25216475- 25216561; the region 4 is chr2: 25216666- 25216752; and the region 5 is chr2: 25216790- 25216899.

[0012] An application according to claim 1, characterized in that the detection product is any one of a reagent, a kit, a chip and a sequencing library.

[0013] Preferably, when the product is a reagent, the detection reagent includes: one or more combinations of nucleic acid combination 1 for detecting the area 1, nucleic acid combination 2 for detecting the area 2, nucleic acid combination 3 for detecting the area 3, nucleic acid combination 4 for detecting the area 4, and nucleic acid combination 5 for detecting the area 5.

[0014] Detection of the methylation status of HCC is explained as: detecting a nucleic acid combination of at least one genomic region or the full-length region or a partial region: region 1, region 2, region 3, region 4 and region 5 can be detected separately; regions 1+2, 1+3, 1+4 can be detected, or regions 2+3+4, 3+4+5 or 1+3+5 can be detected, or the full-length region of region 1+2+3+4+5 can be detected.

[0015] Preferably, the nucleic acid combination 1 includes primer combination 1, the nucleic acid combination 2 includes primer combination 2, the nucleic acid combination 3 includes primer combination 3, the nucleic acid combination 4 includes primer combination 4, and the nucleic acid combination 5 includes primer combination 5;

[0016] The primer combination 1 includes an upstream primer sequence and a downstream primer sequence as shown in SEQ ID NO.1-2;

[0017] The primer combination 2 comprises an upstream primer sequence and a downstream primer sequence as shown in SEQ ID NO.4-5;

[0018] The primer combination 3 comprises an upstream primer sequence and a downstream primer sequence as shown in SEQ ID NO.7-8;

[0019] The primer combination 4 comprises an upstream primer sequence and a downstream primer sequence as shown in SEQ ID NO.10-11;

[0020] The primer combination 5 includes an upstream primer sequence and a downstream primer sequence as shown in SEQ ID NO.13-14.

[0021] Preferably, the nucleic acid combination 1 further includes probe 1, the nucleic acid combination 2 further includes probe 2, the nucleic acid combination 3 further includes probe 3, the nucleic acid combination 4 further includes probe 4, and the nucleic acid combination 5 further includes probe 5;

[0022] The probe 1 has a probe sequence as shown in SEQ ID NO.3;

[0023] The probe 2 has a probe sequence as shown in SEQ ID NO.6;

[0024] The probe 3 has a probe sequence as shown in SEQ ID NO.9;

[0025] The probe 4 has a probe sequence as shown in SEQ ID NO.12;

[0026] The probe 5 has a probe sequence as shown in SEQ ID NO.15.

[0027] The above-mentioned nucleic acid combination includes any one or more combinations of the following nucleic acid combinations: forward / reverse primer nucleic acid combination 1 for detecting the region 1, forward / reverse primer nucleic acid combination 2 for detecting the region 2, forward / reverse primer nucleic acid combination 3 for detecting the region 3, forward / reverse primer nucleic acid combination 4 for detecting the region 4 and forward / reverse primer nucleic acid combination 5 for detecting the region 5.

[0028] The 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a fluorescent quencher group.

[0029] The fluorescent reporter group is 6-HEX, 6-FAM, 6-VIC, 6-TET, 5-CF532, 6-JOE, 5-TAMRA, 6-TAMRA, 6-ROX, CY3, CY5, Texas Red, NED, Alexa Flour or VIC, and the quencher group is MGB, TAMRA, BHQ1, BHQ2, BHQ3 or QSY.

[0030] The reporter group at the 5' end of the probe is 6-FAM, and the quencher group at the 3' end is BHQ-1;

[0031] or the reporter group at the 5' end of the probe is 6-VIC and the quencher group at the 3' end is BHQ-1;

[0032] or the reporter group at the 5' end of the probe is 6-ROX and the quencher group at the 3' end is BHQ-1;

[0033] or the reporter group at the 5' end of the probe is CY5 and the quencher group at the 3' end is BHQ-2;

[0034] Or the reporter group at the 5' end of the probe is 6-HEX, and the quencher group at the 3' end is TAMRA.

[0035] Preferably, the methylation level of the molecular marker is detected by any of the following methods: MSP method, high-throughput sequencing method, methylation-specific high-performance liquid chromatography method, ddPCR method, methylation-specific high-resolution melting curve method, methylation-specific microarray method, methylation-sensitive restriction endonuclease method and Methylight method;

[0036] The high-throughput sequencing method includes bisulfite sequencing, whole genome methylation sequencing or sequencing by synthesis.

[0037] A primary hepatocellular carcinoma detection kit comprises the nucleic acid combination as described above, and also comprises a positive control substance, a negative control substance, a detection primer of an internal reference gene, a detection probe of an internal reference gene, a DNA polymerase and a buffer.

[0038] Preferably, the detection sample of the kit is any one of an ex vivo blood sample, an ex vivo tissue sample and an ex vivo leukocyte sample.

[0039] The in vitro detection samples of the above-mentioned kit are in vitro tissue samples, in vitro blood samples or in vitro ascites samples, and other in vitro body fluid samples such as sputum, saliva, pleural lavage fluid, alveolar lavage fluid and urine; the blood sample is selected from in vitro plasma samples, in vitro serum samples, in vitro whole blood samples or in vitro blood cell samples.

[0040] Compared with the prior art, the present invention has at least the following technical effects:

[0041] The present invention provides an application of a molecular marker methylation level detection reagent in the preparation of a primary hepatocellular carcinoma detection product. The scheme relates to molecular liquid biopsy technology. After research, the inventors found that the methylation level of the CpG site in the chr2:25216400-25216900 region is related to the occurrence of primary hepatocellular carcinoma (HCC). By detecting the methylation level of this region, it can be determined whether the target sample is hepatocellular carcinoma. The above region can be used as a molecular marker for the diagnosis of primary hepatocellular carcinoma. The present invention provides a good molecular marker for the early detection of primary hepatocellular carcinoma (HCC). BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is a research and development flow chart of mining and screening of methylation markers for detecting liver cancer in Example 1;

[0043] Figure 2 This is the ROC diagram of the detection of the methylation marker in Example 3 in the ex vivo blood sample of liver cancer. DETAILED DESCRIPTION

[0044] 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.

[0045] A technical solution of a specific implementation of the present invention is:

[0046] The terms "complementary" and "complementarity" refer to deoxynucleotides / ribonucleotides (e.g., 1 base) or polydeoxynucleotides / ribonucleotides (e.g., base sequences) associated with base pairing rules. For example, the sequence 5'-TAT-3' is complementary to the sequence 3'-ATA-5'. Complementarity can be "partial", in which only some nucleic acid bases are matched according to the base pairing rules. Alternatively, there may be "complete" complementarity between nucleic acids, and the degree of complementarity between nucleic acid chains affects the binding efficiency of hybridization between primers and nucleic acid sequences and between probes and nucleic acid sequences.

[0047] The term "chain polymerase reaction" is used to amplify the target sequence. The method consists of the following steps: adding a large amount of excess primers to the DNA mixture reaction system with the desired target sequence, followed by a precise thermal cycle reaction in the presence of a DNA chain polymerase. Through the principle of "partial" complementarity or "full" complementarity between the primer and the template chain, denaturation-complementary sequence annealing-extension and multiple rounds of repeated thermal reactions are carried out according to the reaction procedure to obtain a high concentration of amplified fragments of the desired target sequence. Due to the repetitive aspect of the method, the method is called "polymerase chain reaction" ("PCR"). Its product is a "PCR product" or "amplicon". Therefore, the present invention includes but is not limited to the nucleic acid detection and determination method of DNA sequencing method, probe hybridization method, methylation-specific high-performance liquid chromatography method, ddPCR method, methylation-specific high-resolution melting curve method, methylation-specific microarray method, methylation-sensitive restriction endonuclease method and Methylight method.

[0048] The term "methylation level" refers to any measured value used to determine the methylation state of one or more CpG dinucleotide sequences within a nucleic acid sequence. The methylation state can optionally be represented by a "methylation level" by comparing the Ct value of the amplification curve after the bisulfite reaction, or by comparing the sequences of bisulfite-treated and untreated nucleic acids. It can be used as a quantitative indicator of methylation levels in multiple copies. The degree of co-methylation is represented or indicated by the methylation state of more than one methylation site. In a methylated region, co-methylation is defined when the methylation state of more than one methylation site is methylated.

[0049] Example 1: Screening process for liver cancer detection methylation markers, such as Figure 1 As shown,

[0050] This embodiment discloses a methylation marker composition for detecting early liver cancer and a screening process thereof.

[0051] In order to discover and screen methylation markers with excellent detection performance, this example first used a customized hybridization capture large panel for targeted methylation sequencing.

[0052] This hybridization capture panel covers 3.98 million CpG methylation sites in the human genome, including most of the genomic regions and genes related to cancer occurrence, with a coverage range 10 times that of traditional DNA methylation chips.

[0053] By performing targeted sequencing on 82 clinical frozen tissue samples and 115 ex vivo blood samples and comparing the changes in methylation patterns between cancer and non-cancer groups, this example preliminarily identified 30,008 methylation sites that can clearly distinguish cancer and non-cancer samples in both tissue and blood samples as potential cancer markers.

[0054] Furthermore, a hybrid capture panel was designed based on the 30,008 methylation sites initially screened above for the 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 calculation and facilitated the accurate screening of markers in the second round.

[0055] By performing targeted methylation sequencing on ex vivo plasma samples from 218 clinical liver cancer patients and 196 ex vivo plasma samples from non-cancer groups, univariate statistical analysis and machine learning methods were used to accurately screen out 12 DNA methylation markers with the best performance for liver cancer detection.

[0056] Finally, primer and probe nucleic acid combinations of the qPCR detection platform were designed for the genomic regions of the 12 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.

[0057] Finally, the DNA methylation marker with the best performance for liver cancer detection was determined through univariate performance analysis, namely the methylation level of the chr2:25216400-25216900 genomic region.

[0058] The primer combination for detecting the methylation level of the marker includes any one of SEQ ID NO.1-2, SEQ ID NO.4-5, SEQ ID NO.7-8, SEQ ID NO.10-11 and SEQ ID NO.13-14.

[0059] The nucleic acid probe used for detection includes any one of SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12 and SEQ ID NO.15.

[0060] Example 2: Detection performance of the screened markers in liver cancer tissue samples

[0061] 33 clinical samples of liver cancer tissue and 49 non-cancer control tissue samples were collected. The tissue samples were subjected to genome extraction, enzyme-based methylation conversion, and targeted methylation sequencing. The specific experimental steps are as follows:

[0062] 1. DNA sample extraction: When the sample is a frozen fresh tissue sample, use QIAGEN's DNeasy ®The genome was extracted using Blood&Tissue Kit #69506. For specific operations, please refer to the kit instructions. Genomic DNA was sheared using a Covaris M220 ultrasonic shearer. When cfDNA was extracted from plasma samples, Guangzhou Youze Biotechnology's Free DNA Extraction Kit (Filtration Method) #C02-1 was used. For specific operations, please refer to the kit instructions.

[0063] 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.

[0064] 3. Quality control preparation: 10 µL of 0.1 ng / µL CpG methylated pUC19 and 10 µL of 2 ng / µL unmethylated λ DNA should be added to each sample;

[0065] 4. Library construction for methylation sequencing: Use the NEBNext Enzymatic Methyl-Seq kit to construct the library.

[0066] 4.1 End repair and 3' end addition of "A"

[0067] 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.

[0068]

[0069] 4.1.2 Place in PCR instrument and perform reaction according to the following procedure

[0070]

[0071] 4.2 Connector connection

[0072] 4.2.1 Add the following reagents to the end repair product for reaction

[0073]

[0074] 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.

[0075] 4.2.3 Purification of adapter ligation products

[0076] A. Equilibrate the purified magnetic beads at room temperature for 30 minutes and mix them thoroughly on a vortex.

[0077] B. Prepare 80% ethanol according to the ratio of anhydrous ethanol to nuclease-free water = 8:2 and set aside.

[0078] 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.

[0079] 4.3 Methylation: Oxidation reaction

[0080] 4.3.1 Buffer configuration:

[0081] 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.

[0082] 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.

[0083] c Dilute the Stop Buffer 1:10 with nuclease-free water.

[0084] 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.

[0085]

[0086] 4.3.3 Place the tube in a PCR instrument and react under the following conditions: 37°C, 1h, heating cover temperature ≥ 45°C.

[0087] 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.

[0088] 4.3.5 Purification of oxidation reaction products

[0089] A. Equilibrate the purified magnetic beads at room temperature for 30 minutes and thoroughly mix them on a vortex machine;

[0090] B. Prepare 80% ethanol in a ratio of 8:2 for absolute ethanol and nuclease-free water, and set aside.

[0091] 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.

[0092] 4.4 Methylation: cytosine deamination

[0093] 4.4.1 Denaturation:

[0094] a. Preheat the PCR instrument to 85°C and open the heated cover;

[0095] b Add 2 μL of formamide to 8 μL of the purified oxidation reaction product, vortex to mix, and centrifuge briefly.

[0096] 4.4.2 Add the following reagents to the denatured product for reaction

[0097]

[0098] 4.4.3 Place in PCR instrument and react under the following conditions: 37℃, 3h, heating cover temperature ≥ 45℃.

[0099] 4.4.4 Purification of Cytosine Deamination Reaction Products

[0100] a. Equilibrate the purified magnetic beads at room temperature for 30 minutes and thoroughly mix them on a vortex machine;

[0101] b Prepare 80% ethanol according to the ratio of anhydrous ethanol to nuclease-free water = 8:2 and set aside;

[0102] 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.

[0103] 4.5 PCR amplification and purification

[0104] 4.5.1 Add the following reagents to the above purified product for reaction

[0105]

[0106] 4.5.2 Place on PCR instrument and react according to the following conditions

[0107]

[0108] 4.5.3 Purification of PCR amplification products

[0109] a. Equilibrate the purified magnetic beads at room temperature for 30 minutes and thoroughly mix them on a vortex machine;

[0110] b Prepare 80% ethanol in the ratio of anhydrous ethanol to nuclease-free water = 8:2 and set aside;

[0111] 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.

[0112] 5. Hybridization and elution

[0113] 5.1 Hybridization:

[0114] a Take 10 ng of each library to be hybridized, calculate the library volume required for combined hybridization, and mix the 12 libraries together;

[0115] b. Add the following reagents to the library mixture for reaction;

[0116] c Turn on the vacuum concentrator, set the V-AQ mode, and concentrate into dry powder at room temperature;

[0117] 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;

[0118] e Place on a PCR instrument and perform the reaction under the following conditions.

[0119] Reagent composition table

[0120]

[0121] PCR reaction conditions table

[0122]

[0123] 5.2 Capture and elution

[0124] 5.2.1 Buffer preheating:

[0125] 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.

[0126] 5.2.2 Magnetic bead capture

[0127] a Vortex to mix the streptavidin magnetic beads that have been equilibrated to room temperature;

[0128] b. Take 70 μL of streptavidin magnetic beads and add them into a 1.5 mL centrifuge tube;

[0129] 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;

[0130] d. Repeat steps b and c twice, performing streptavidin magnetic bead washing three times in total;

[0131] e Add 20 μL binding buffer to resuspend the streptavidin magnetic beads;

[0132] 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.

[0133] g. Mix on a mixer at room temperature for 30 minutes without vortexing.

[0134] h After mixing, centrifuge immediately, place on a magnetic stand, let stand for 1 minute, and discard the supernatant.

[0135] 5.2.3 Elution

[0136] 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;

[0137] 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;

[0138] c Place on a magnetic stand, let stand for 1 minute, and discard the supernatant;

[0139] 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.

[0140] e After incubation, centrifuge immediately, place on a magnetic stand, let stand for 1 min, and discard the supernatant;

[0141] 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;

[0142] g After the incubation is completed, centrifuge briefly, place on a magnetic stand, let stand for 1 minute, and discard the supernatant;

[0143] h. Repeat steps a to g twice for a total of three washes;

[0144] 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.

[0145] 5.3 PCR enrichment of capture products

[0146] 5.3.1 Take 22.5 μL of the captured product (streptavidin magnetic bead suspension) and add the following reagents to react.

[0147]

[0148] Place on PCR instrument and react according to the following conditions

[0149]

[0150] 5.3.2 PCR product purification

[0151] a. Equilibrate the DNA purification magnetic beads at room temperature for 30 minutes and mix thoroughly on a vortex machine;

[0152] b Prepare 80% ethanol in the ratio of anhydrous ethanol to nuclease-free water = 8:2 and set aside;

[0153] 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.

[0154] 6. Analysis of sequencing data:

[0155] 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.

[0156] The detection performance of the determined methylation markers and the combination for distinguishing liver cancer tissues from non-cancer tissues is shown in the following table:

[0157]

[0158] Example 3: Detection performance of the screened markers in liver cancer blood samples, such as Figure 2 As shown,

[0159] 414 clinical blood samples were collected, including 218 blood samples from patients with primary liver cancer, 173 blood samples from patients with benign liver diseases, and 23 blood samples from healthy people. The blood samples were subjected to free DNA extraction, methylation conversion, and targeted methylation sequencing according to the method described in Example 2, and the methylation level of each methylation marker was calculated.

[0160] Blood sample statistical results:

[0161]

[0162] Single factor statistical analysis was performed on single methylation markers to calculate the detection performance of ex vivo blood samples for distinguishing liver cancer patients from controls. The sensitivity and specificity of liver cancer detection of the determined methylation markers are shown in the following table:

[0163]

[0164] like Figure 2 As shown, the AUC value of the determined methylation marker for methylation sequencing detection of liver cancer ex vivo blood samples was 0.933.

[0165] Example 4: Performance of the identified methylation markers in an ex vivo blood sample qPCR detection dataset for liver cancer.

[0166] This embodiment provides a primary liver cancer diagnostic reagent, which specifically includes the following nucleic acid combinations for detecting CpG methylation in the chr6:26250425-26250945 gene region: nucleic acid combination 1, nucleic acid combination 2, nucleic acid combination 3, nucleic acid combination 4 and nucleic acid combination 5.

[0167] Region 1: chr2: 25216400- 25216504, primer probe nucleic acid combination 1 is as follows:

[0168] F-01: 5'-CGGGGTCGTTTTTGTTT-3' (SEQ ID NO.1)

[0169] R-01: 5'-CGACGCAATTATCACCG-3' (SEQ ID NO.2)

[0170] P-01: 5'-TATAACGTATTCCTTCACTCATCGAAAT-3' (SEQ ID NO.3)

[0171] Region 2: chr2: 25216454- 25216521, primer probe nucleic acid combination 2 is as follows:

[0172] F-02: 5'-CGAGTTATCGCGTTCGTC-3' (SEQ ID NO.4)

[0173] R-02: 5'-AAAAACACGCTATAATACTCACTC-3' (SEQ ID NO.5)

[0174] P-02: 5'-CACTCAATACAACATCGATAATAATTACG-3' (SEQ ID NO.6)

[0175] Region 3: chr2: 25216475- 25216561, primer probe nucleic acid combination 3 is as follows:

[0176] F-03: 5'-TTTAATGTAGTATCGGTGATAATTG-3' (SEQ ID NO.7)

[0177] R-03: 5'-CCTCCCTCCTCGTCCTCAAAAATA-3' (SEQ ID NO.8)

[0178] P-03: 5'-AACGCGAACCACCGCG-3' (SEQ ID NO.9)

[0179] Region 4: chr2: 25216666- 25216752, primer probe nucleic acid combination 4 is as follows:

[0180] F-04: 5'-AGACGGGTTTTAATTTTGTTGT-3' (SEQ ID NO.10)

[0181] R-04: 5'-TCGCTTAAATCTAAAAAACGA-3' (SEQ ID NO. 11)

[0182] P-04: 5'-GAAATCGCGCCATTACACCG-3' (SEQ ID NO. 12)

[0183] Region 5: chr2: 25216790- 25216899, primer probe nucleic acid combination 5 is as follows:

[0184] F-04: 5'-GGTATGCGTTATAATGTTTAATTAA-3' (SEQ ID NO.13)

[0185] R-04: 5'-CGAAACGAACGAATAACCTAA-3' (SEQ ID NO.14)

[0186] P-04: 5'-AAAATTCGAAATAAACCTAACCAACAT-3' (SEQ ID NO.15)

[0187] The primer probes for the internal reference gene are as follows:

[0188] b-actin-F01: 5'-GTGATGGAGGAGGTTTAGTAAGTT-3' (SEQ ID NO. 16)

[0189] b-actin-R01: 5'-ACCACCACCCAACACACAATAACAAACACA-3' (SEQ ID NO.17)

[0190] b-actin-P01: 5'-CCAATAAAAACCTACTCCTCCCTTAA-3' (SEQ ID NO. 18)

[0191] A total of 79 clinical blood samples were collected in this experiment, including 32 blood samples from patients with primary liver cancer, 32 blood samples from patients with benign liver diseases, and 15 blood samples from healthy people.

[0192]

[0193] The 79 blood samples collected above were subjected to quantitative methylation qPCR detection, and the qPCR detection Ct values ​​were recorded. The specific experimental steps are as follows:

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198]

[0199] 5. Reaction system for methylation quantitative PCR:

[0200] 6. Reaction procedure of methylation quantitative PCR:

[0201]

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] The Ct values ​​of qPCR detection using the designed primer-probe combination in ex vivo blood samples were used to distinguish liver cancer and non-cancer samples. The detection performance is shown in the following table:

[0207]

[0208] From the blood qPCR test results, it can be seen that the liver cancer detection performance of different primer probe combinations designed for the chr2:25216400-25216900 marker is similar, with a specificity of 89.4-91.5% and a sensitivity of 81.3-84.4%. Compared with traditional liver cancer screening methods, this single methylation marker has ideal detection performance, is easy to use and has low cost, which is of great significance for the popularization and promotion of early screening and treatment of liver cancer.

[0209] 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. Use of a molecular marker methylation level detection reagent in the preparation of a primary hepatocellular carcinoma detection product, characterized in that: The molecular marker is a nucleic acid molecule containing at least one CpG site in the chr2:25216400-25216900 genomic region sequence; The genomic region is from the hg38 human reference genome; The molecular marker is located in the following regions containing at least one CpG site: region 1, region 2, region 3, region 4, and region 5; The region 1 is chr2: 25216400- 25216504; the region 2 is chr2: 25216454- 25216521; the region 3 is chr2: 25216475- 25216561; the region 4 is chr2: 25216666- 25216752; and the region 5 is chr2: 25216790- 25216899.

2. An application according to claim 1, characterized in that: The detection product is any one of a reagent, a kit, a chip and a sequencing library.

3. An application according to claim 1, characterized in that: When the product is a reagent, the detection reagent includes: one or more combinations of nucleic acid combination 1 for detecting the area 1, nucleic acid combination 2 for detecting the area 2, nucleic acid combination 3 for detecting the area 3, nucleic acid combination 4 for detecting the area 4, and nucleic acid combination 5 for detecting the area 5.

4. An application according to claim 3, characterized in that: The nucleic acid combination 1 includes a primer combination 1, the nucleic acid combination 2 includes a primer combination 2, the nucleic acid combination 3 includes a primer combination 3, the nucleic acid combination 4 includes a primer combination 4, and the nucleic acid combination 5 includes a primer combination 5; The primer combination 1 includes an upstream primer sequence and a downstream primer sequence as shown in SEQ ID NO.1-2; The primer combination 2 comprises an upstream primer sequence and a downstream primer sequence as shown in SEQ ID NO.4-5; The primer combination 3 comprises an upstream primer sequence and a downstream primer sequence as shown in SEQ ID NO.7-8; The primer combination 4 comprises an upstream primer sequence and a downstream primer sequence as shown in SEQ ID NO.10-11; The primer combination 5 includes an upstream primer sequence and a downstream primer sequence as shown in SEQ ID NO.13-14.

5. A use according to claim 4, characterized in that: The nucleic acid combination 1 further includes probe 1, the nucleic acid combination 2 further includes probe 2, the nucleic acid combination 3 further includes probe 3, the nucleic acid combination 4 further includes probe 4, and the nucleic acid combination 5 further includes probe 5; The probe 1 has a probe sequence as shown in SEQ ID NO.3; The probe 2 has a probe sequence as shown in SEQ ID NO.6; The probe 3 has a probe sequence as shown in SEQ ID NO.9; The probe 4 has a probe sequence as shown in SEQ ID NO.12; The probe 5 has a probe sequence as shown in SEQ ID NO.

15.

6. The use according to claim 1, characterized in that: The methylation level of the molecular marker is detected by any of the following methods: MSP method, high-throughput sequencing method, methylation-specific high-performance liquid chromatography method, ddPCR method, methylation-specific high-resolution melting curve method, methylation-specific microarray method, methylation-sensitive restriction endonuclease method and Methylight method; The high-throughput sequencing method includes bisulfite sequencing, whole genome methylation sequencing or sequencing by synthesis.

7. A primary hepatocellular carcinoma detection kit, characterized in that: The method comprises the nucleic acid combination as claimed in claim 3, and further comprises 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 primary hepatocellular carcinoma detection kit according to claim 7, characterized in that: The detection sample of the kit is any one of an ex vivo blood sample, an ex vivo tissue sample and an ex vivo leukocyte sample.

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