Application of a dual liver cancer molecular marker in the preparation of a liver cancer detection reagent product

Abstract

The present invention discloses the application of a dual liver cancer molecular marker in the preparation of liver cancer detection reagent products, belonging to the technical field of detection kits. The dual liver cancer molecular marker is a composition of DNA methylation dual liver cancer molecular markers screened by the inventor from human genome regions covering more than 10 times the 450K chip range through two rounds of targeted methylation sequencing and suitable for early liver cancer detection. In the sample dataset used in the present invention, the detected performance of the screened markers and composition is optimal, which can further improve the detection accuracy on the basis of a single methylation marker and avoid the problems of false detection and missed detection. Compared with the commonly used liver cancer screening methods, the dual liver cancer molecular marker has significantly improved stability, sensitivity and accuracy, and can be used for the detection of in vitro blood samples, which is very beneficial to the popularization and promotion of early screening and early diagnosis of liver cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection kits, and particularly relates to the application of a double hepatocellular carcinoma molecular marker in the preparation of a hepatocellular carcinoma detection reagent product. Background Art

[0002] "Early screening, early diagnosis and early treatment" of hepatocellular carcinoma is considered to be one of the important means to reduce the impact of hepatocellular carcinoma on patients and improve the survival rate of patients. Especially for small hepatocellular carcinomas within 5 cm, the 5-year survival rate of patients treated early can reach more than 80%. However, the current commonly used hepatocellular carcinoma screening methods, AFP and ultrasound examination, have unsatisfactory sensitivity, resulting in a relatively serious problem of missed detection. Therefore, developing a more accurate and convenient hepatocellular carcinoma screening method is of great significance for the popularization and promotion of early screening of hepatocellular carcinoma.

[0003] Abnormal DNA methylation signals are one of the characteristics of cancer occurrence. In recent years, studies have shown that the abnormal methylation signals carried by cell-free DNA in blood have good stability and can be used as molecular markers to detect the occurrence of cancer. Compared with blood AFP levels, cell-free DNA methylation markers are more sensitive and specific for the detection of hepatocellular carcinoma. Although some cell-free DNA methylation markers for hepatocellular carcinoma have been preliminarily reported, most of these markers are screened from 450K methylation chip data, covering less than 2% of the human genome methylation sites.

[0004] Therefore, using a more comprehensive marker screening strategy and method is the key to developing more accurate hepatocellular carcinoma methylation markers. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of a double hepatocellular carcinoma molecular marker in the preparation of a hepatocellular carcinoma detection reagent product. The inventors screened out a composition of DNA methylation double hepatocellular carcinoma molecular markers suitable for early hepatocellular carcinoma detection from human genome regions covering more than 10 times the 450K chip coverage through two rounds of targeted methylation sequencing. The double hepatocellular carcinoma molecular marker has significantly improved stability, sensitivity and accuracy compared with the commonly used hepatocellular carcinoma screening methods, and can be used for the detection of in vitro blood samples, which is very beneficial for the popularization and promotion of early screening and early diagnosis of hepatocellular carcinoma.

[0006] The present invention is achieved through the following technical solutions:

[0007] The application of a double hepatocellular carcinoma molecular marker in the preparation of a hepatocellular carcinoma detection reagent product, wherein the double hepatocellular carcinoma molecular marker is the CpG methylation sites in the genomic regions of chr2:25216400-25216752 and chr6:26250469-26250942;

[0008] The genomic regions belong to the hg38 human genome.

[0009] Preferably, the reagent product includes any one of reagents, reagent kits, chips, hybridization probes, and sequencing libraries.

[0010] A dual-nucleic acid combination for detecting liver cancer, comprising nucleic acid combination A for detecting the CpG methylation level in the genomic region of chr2:25216400-25216752;

[0011] and nucleic acid combination B for detecting the CpG methylation level in the genomic region of chr6:26250469-26250942.

[0012] Preferably, the nucleic acid combination A includes a first primer pair combination and a first probe;

[0013] The base sequence of the first primer pair combination has at least 90% similarity with the base sequences shown in sequences 1-2, sequences 4-5, or sequences 7-8;

[0014] The base sequence of the first probe has at least 90% similarity with the base sequences shown in sequences 3, sequences 6, or sequences 9.

[0015] Preferably, the nucleic acid combination B includes a second primer pair combination and a second probe;

[0016] The base sequence of the second primer pair combination has at least 90% similarity with the base sequences shown in sequences 10-11, sequences 13-14, sequences 16-17, or sequences 19-20;

[0017] The base sequence of the second probe has at least 90% similarity with the base sequences shown in sequences 12, sequences 15, sequences 18, or sequences 21.

[0018] A reagent kit for detecting liver cancer, the reagent kit containing the dual liver cancer molecular markers or the dual-nucleic acid combination for detecting liver cancer as described above.

[0019] Preferably, the methylation level in the reagent kit is detected by any one of the following methods: methylation-specific PCR method, methylation-specific microarray method, methylation sequencing method, methylation-specific high-performance liquid chromatography method, digital PCR method, methylation-specific high-resolution melting curve method.

[0020] Preferably, the samples detected in the reagent kit include any one of ex vivo tissue samples, ex vivo plasma samples, ex vivo serum samples, ex vivo whole blood samples, ex vivo blood cell samples, and urine samples.

[0021] Compared with the prior art, the present invention has at least the following technical effects:

[0022] 1. The present invention provides an application of a dual liver cancer molecular marker in the preparation of a liver cancer detection reagent product. The inventors screened out a composition of DNA methylation dual liver cancer molecular markers suitable for early liver cancer detection from human genomic regions covering more than 10 times the 450K chip range through two rounds of targeted methylation sequencing. In the sample dataset used in the present invention, the detection performance of the screened markers and composition is optimal. Compared with common liver cancer screening methods, the dual liver cancer molecular marker has significantly improved stability, sensitivity, and accuracy, and can be used for the detection of in vitro blood samples, which is very beneficial for the popularization and promotion of early screening and diagnosis of liver cancer.

[0023] 2. The kit for detecting liver cancer contains two DNA methylation markers, and the composition of the dual liver cancer molecular markers is related to the occurrence of liver cancer. Through the combined results of the methylation levels of the two markers, it is suitable for assisting doctors in judging whether early liver cancer has occurred.

[0024] 3. The kit uses DNA methylation dual liver cancer molecular markers. Compared with traditional tumor markers or liver cancer detection methods, the DNA methylation dual liver cancer molecular markers have high detection sensitivity, more stable detection results, and can be detected at an early stage of cancer development, which is beneficial for the discovery of liver cancer in the initial stage of development. The kit uses a composition of methylation dual liver cancer molecular markers, which can further improve the detection accuracy on the basis of a single methylation marker and avoid the problems of false detection and missed detection.

[0025] 4. In the screening process of the dual liver cancer molecular marker, the methylation sequencing adopted is based on the enzyme conversion method for the conversion of cytosine to uracil. Compared with the traditional bisulfite conversion, it can avoid damage to DNA, thus solving the problem of low content of cell-free DNA in blood. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a R & D flow chart for the discovery and screening of methylation dual liver cancer molecular markers for detecting liver cancer in Example 1;

[0027] Figure 2 It is an ROC graph for the detection of methylation dual liver cancer molecular marker composition in in vitro blood samples of liver cancer in Example 3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] 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.

[0029] Example 1: The screening process of methylation dual liver cancer molecular markers for liver cancer detection. The specific process is as Figure 1 shown.

[0030] This example discloses a methylation dual liver cancer molecular marker composition for detecting early liver cancer and its screening process.

[0031] In order to discover and screen methylation dual liver cancer molecular markers with excellent detection performance, this example first adopted a customized hybridization capture large panel for targeted methylation sequencing.

[0032] This hybridization capture panel covers 3.98 million CpG methylation sites in the human genome, which covers the vast majority of CpG island regions and genes related to cancer occurrence, and the coverage range is 10 times that of traditional DNA methylation chips.

[0033] 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 identified 30,008 methylation sites that can significantly distinguish cancer samples from non-cancer samples in both tissue samples and blood samples as potential cancer markers.

[0034] According to the above 30,008 initially screened methylation sites, a hybridization capture panel was designed 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 the second-round markers. By performing targeted methylation sequencing on 218 ex vivo plasma samples of clinical liver cancer patients and 196 ex vivo plasma samples of the non-cancer group, and using univariate statistical analysis and machine learning methods, 12 DNA methylation markers with the best liver cancer detection performance were precisely screened out.

[0035] Finally, primers and probe nucleic acid combinations for the qPCR detection platform were designed for the genomic regions of the 12 screened methylation markers, and the performance verification of liver cancer detection was carried out on the qPCR platform using 32 ex vivo plasma samples of clinical liver cancer patients and 47 ex vivo plasma samples of the non-cancer group.

[0036] Finally, through an exhaustive combination method, the combination of the dual DNA methylation dual liver cancer molecular markers with the best liver cancer detection performance was determined, that is, the methylation levels of the genomic regions chr2:25216400 - 25216752 and chr6:26250469 - 26250942.

[0037] The first primer pair combination for detecting the methylation level of the double biomarker composition includes any one of sequences 1-2, sequences 4-5, or sequences 7-8; the second primer pair combination includes any one of sequences 10-11, sequences 13-14, sequences 16-17, or sequences 19-20.

[0038] The first nucleic acid probe for detection includes any one of sequences 3, 6, or 9; the second probe sequence includes any one of sequences 12, 15, 18, or 21.

[0039] Example 2: Detection performance of the determined methylated double liver cancer molecular markers in the methylated sequencing dataset of liver cancer tissue samples

[0040] Targeted methylation sequencing was performed using 33 collected clinical liver cancer tissue samples and 49 non-cancer control tissue samples.

[0041] The specific experimental steps are as follows:

[0042] 1. DNA sample extraction: When the sample is a frozen fresh tissue sample, the genomic DNA was extracted using the "DNeasy® Blood&Tissue Kit #69506" from QIAGEN. The specific operation refers to the kit instructions. The genomic DNA was fragmented using a Covaris M220 ultrasonic disruptor. When extracting cfDNA from plasma samples, the "Free DNA Extraction Kit (Filtration Method) #C02-1" from Guangzhou Youze Biotechnology was used. The specific operation refers to the kit instructions.

[0043] 2. Fragment screening of the extracted DNA samples using SPRIselect magnetic beads according to the recommended steps in the instructions, filtering out DNA molecules larger than 200 bp.

[0044] 3. Preparation of quality control products: 10 µL of 0.1 ng / µL CpG-methylated pUC19 and 10 µL of 2 ng / µL non-methylated λDNA were added to each sample.

[0045] 4. Library construction for methylation sequencing: The NEBNext Enzymatic Methyl-Seq kit was used for library construction.

[0046] 4.1 End repair and addition of "A" at the 3' end

[0047] 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.

[0048]

[0049] 4.1.2 Place it in a PCR instrument and perform the reaction according to the following procedure

[0050]

[0051] 4.2 Ligation of adapters

[0052] 4.2.1 Add the following reagents to the end-repaired product for reaction

[0053]

[0054] 4.2.2 Place it in a PCR instrument and set the PCR reaction program according to the following conditions: 60 °C, 60 min, hot lid closed.

[0055] 4.2.3 Purification of the adapter-ligated product

[0056] A. Equilibrate the purification magnetic beads at room temperature for 30 min and mix the purification magnetic beads thoroughly on a vortex mixer.

[0057] B. Prepare 80% ethanol according to the ratio of absolute ethanol: nuclease-free water = 8:2 for later use.

[0058] C. Add 55 μL of 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, and pipette 14 μL of the supernatant for the next reaction.

[0059] 4.3 Methylation: Oxidation reaction

[0060] 4.3.1 Prepare the buffer

[0061] a. Add 400 μL of E1 reaction buffer supplement to 100 μL of E1 reaction buffer, mix well by oscillation, and mark the preparation date.

[0062] 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, prepared freshly and not stored.

[0063] c. Dilute the stop buffer with nuclease-free water at a ratio of 1:10.

[0064] 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 by centrifugation.

[0065]

[0066] 4.3.3 Place it on a PCR instrument and react under the following conditions: 37°C, 1 h, and the hot lid temperature ≥ 45°C.

[0067] 4.3.4 After the reaction is completed, add 1 μL of the diluted reaction termination solution to the product, place it on a PCR instrument, and react under the following conditions: 37°C, 30 min, and the hot lid temperature ≥ 45°C.

[0068] 4.3.5 Purification of the oxidation reaction product

[0069] A. Equilibrate the purification magnetic beads at room temperature for 30 min and mix the purification magnetic beads thoroughly on a vortex mixer;

[0070] B. Prepare 80% ethanol according to the ratio of absolute ethanol: nuclease-free water = 8:2 and set aside;

[0071] 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 it on a magnetic rack, and discard the supernatant after the solution becomes clear; wash with the freshly prepared 80% ethanol and discard the supernatant, and repeat this step once; dry, elute with 9.5 μL of elution buffer, and pipette 8 μL of the supernatant for the next reaction.

[0072] 4.4 Methylation: Cytosine deamination

[0073] 4.4.1 Denaturation:

[0074] a Preheat the PCR instrument to 85°C and turn on the hot lid;

[0075] b Add 2 μL of formamide to 8 μL of the purified oxidation reaction product, vortex and mix well, and centrifuge briefly.

[0076] 4.4.2 Add the following reagents to the denatured product for reaction

[0077]

[0078] 4.4.3 Place it on a PCR instrument and react under the following conditions: 37°C, 3 h, and the hot lid temperature ≥ 45°C.

[0079] 4.4.4 Purification of the cytosine deamination reaction product

[0080] a Equilibrate the purification magnetic beads at room temperature for 30 min and mix the purification magnetic beads thoroughly on a vortex mixer;

[0081] b Prepare 80% ethanol according to the ratio of absolute ethanol: nuclease-free water = 8:2 and set aside;

[0082] c Add 50 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 11 μL of elution buffer, and pipette 10 μL of the supernatant for the next reaction.

[0083] 4.5 PCR Amplification and Purification

[0084] 4.5.1 Add the following reagents to the above purified product for reaction

[0085]

[0086] 4.5.2 Place on a PCR instrument and react under the following conditions

[0087]

[0088] 4.5.3 Purification of PCR Amplification Product

[0089] a Equilibrate the purification magnetic beads at room temperature for 30 min and mix well on a vortex mixer;

[0090] b Prepare 80% ethanol by mixing absolute ethanol and nuclease-free water in a ratio of 8:2 and set aside;

[0091] 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, and pipette 14 μL of the supernatant for quality inspection.

[0092] 5. Hybridization and Elution

[0093] 5.1 Hybridization:

[0094] 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;

[0095] b Add the following reagents to the library mixture for reaction;

[0096] c Turn on the vacuum concentrator, set the V-AQ mode, and concentrate into dry powder at room temperature;

[0097] 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;

[0098] e Place on a PCR instrument and react under the following conditions.

[0099] Reagent Component Table

[0100]

[0101] PCR Reaction Condition Table

[0102]

[0103] 5.2 Capture and Elution

[0104] 5.2.1 Buffer Preheating:

[0105] Preheat the rapid binding solution and rapid washing solution 2 to dissolve the precipitate at 48°C, preheat the rapid washing solution 1 to dissolve the precipitate at 63°C, and equilibrate the streptavidin magnetic beads at room temperature for at least 30 min.

[0106] 5.2.2 Magnetic Bead Capture

[0107] a Vortex and mix the streptavidin magnetic beads that have been equilibrated to room temperature;

[0108] b Take 70 μL of streptavidin magnetic beads and add them to a 1.5 mL centrifuge tube;

[0109] 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;

[0110] d Repeat steps b and c twice for a total of three washes of the streptavidin magnetic beads;

[0111] e Resuspend the streptavidin magnetic beads with 20 μL of binding buffer;

[0112] 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.

[0113] g Mix on a mixer for 30 minutes at room temperature without vortexing;

[0114] h After mixing, centrifuge briefly, place on a magnetic stand, let stand for 1 minute, and discard the supernatant.

[0115] 5.2.3 Elution

[0116] a Add 200 μL of rapid washing buffer 1 preheated to 54°C, pipette and mix well, and incubate at 54°C in a thermostatic metal bath for 5 min;

[0117] 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;

[0118] c Place it on a magnetic stand, let it stand for 1 minute, and discard the supernatant;

[0119] d Add 200 μL of washing buffer 1 preheated to 63 °C, pipette and mix well, and incubate in a thermostatic metal bath at 54 °C for 5 minutes;

[0120] e After incubation, centrifuge briefly, place it on a magnetic stand, let it stand for 1 min, and discard the supernatant;

[0121] f Add 200 μL of washing buffer 2 preheated to 54 °C, pipette and mix well, and incubate in a thermostatic metal bath at 54 °C for 5 minutes;

[0122] g After incubation, centrifuge briefly, place it on a magnetic stand, let it stand for 1 minute, and discard the supernatant;

[0123] h Repeat steps a to g twice for a total of three washes;

[0124] i Centrifuge briefly, use a 10 μL pipette tip to discard the remaining supernatant, immediately add 45 μL of nuclease-free water, pipette and mix well, and incubate on ice.

[0125] 5.3 PCR enrichment of the captured product

[0126] 5.3.1 Take 22.5 μL of the captured product (streptavidin magnetic bead suspension) and add the following reagents for reaction.

[0127]

[0128] Place it on a PCR instrument and react under the following conditions

[0129]

[0130] 5.3.2 Purification of the PCR product

[0131] a Equilibrate the DNA purification magnetic beads at room temperature for 30 min and mix well on a vortex mixer;

[0132] b Prepare 80% ethanol by mixing absolute ethanol and nuclease-free water in a ratio of 8:2 for later use;

[0133] 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 it on a magnetic stand, and discard the supernatant after the solution becomes clear; wash with the newly 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 a machine.

[0134] 6. Analysis of sequencing data:

[0135] The original data from the sequencer is first filtered using fastQC to remove low-quality sequences (phred33 score ≤ 20), as well as 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 regions (regions corresponding to DNA methylation markers) are selected using samtools, and the methylation levels of individual DNA methylation markers are calculated.

[0136] The detection performance of the identified methylation markers in distinguishing liver cancer tissues from non-cancerous tissues is as follows in the table,

[0137]

[0138] Example 3: Performance of the identified methylation markers in the methylation sequencing dataset of ex vivo blood samples from liver cancer patients

[0139] Targeted methylation sequencing was performed using ex vivo blood samples from 218 clinical liver cancer patients and 196 non-cancer control groups that were collected, and the methylation levels of each methylation marker were calculated. Univariate statistical analysis was performed on a single methylation marker to calculate the detection performance in distinguishing ex vivo blood samples of liver cancer patients from the control group. Combining the identified double markers of chr2:25216400-25216752 and chr6:26250469-26250942 for liver cancer prediction, the detection performance of the double marker combination was statistically analyzed.

[0140] As Figure 2 shown, the AUC value of the identified methylation marker combination for methylation sequencing detection of ex vivo blood samples from liver cancer patients is 0.951; the detection sensitivity and specificity are as follows in the table:

[0141] Example 4: Performance of the identified methylation markers in the qPCR detection dataset of ex vivo blood samples from liver cancer patients

[0142] Primer-probe combinations required for the methylation qPCR detection platform were designed respectively for the identified double markers of chr2:25216400-25216752 and chr6:26250469-26250942, and the design results are as follows:

[0143]

[0144] Methylation qPCR detection was performed using ex vivo blood samples from 32 clinical liver cancer patients and 47 non-cancer control groups that were collected, and the Ct values of the qPCR detection were recorded. The specific experimental steps are as follows:

[0145] 1. Plasma Separation: First, centrifuge the collected 10 mL 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 separating impurities should be stored in a -80°C refrigerator for future use.

[0146] 2. Free DNA Extraction: Use the "Free DNA Extraction Kit" (product number: C03-2) from Guangzhou Youze Biotechnology Co., Ltd. to extract plasma free DNA from the separated plasma sample.

[0147] 3. Free DNA Conversion: Use the "EZ-96 DNA Methvlation-LightningMagPrep" from ZYMO to perform methylation C->T conversion on the extracted free DNA sample.

[0148] 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. All C's in the negative control become T after conversion. 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, the C at the methylated CpG positions does not change, while the C at other positions becomes T.

[0149] 5. Reaction System for Methylation Quantitative PCR:

[0150]

[0151] 6. Reaction Program for Methylation Quantitative PCR:

[0152]

[0153] 7. Instrument Detection: Mix the DNA of the sample to be detected, negative reference, and 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 for detection, and collect the fluorescence values of each channel of HEX / ROX / FAM / CY5.

[0154] 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.

[0155] 9. Quality Control: During each test, negative and positive references are detected synchronously. The negative control is the genomic DNA of transformed cells (cell line: QSG-7701) containing the β-actin gene and the hypomethylated target gene sequence, and the positive control is the cfDNA of transformed cells (cell line: HepG2) containing the β-actin gene and the hypermethylated target gene sequence, 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, the current 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.

[0156] 10. Result Determination: If the Ct value of the target marker is less than 35, the test result is positive; otherwise, it 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, it is negative.

[0157] The CT values obtained from qPCR detection of the designed primer-probe combination in ex vivo blood samples are used to distinguish liver cancer and non-cancer samples. The detection performance is as follows in the table:

[0158]

[0159] It can be seen from the plasma qPCR test results that the detection sensitivity for ex vivo blood samples of liver cancer using the methylation markers of chr2:25216400-25216752 or chr6:26250469-26250942 is 81.3% to 84.4%, and the specificity is about 90%. When combining double methylation markers, the detection sensitivity can be increased to 87.5 - 93.8%, and the specificity remains at about 90%.

[0160] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used 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. Use of a dual liver cancer molecular marker detection reagent in the preparation of a liver cancer detection reagent product, characterized in that: The dual liver cancer molecular markers are CpG methylation sites in the genomic regions of chr2:25216400-25216752 and chr6:26250469-26250942; The genomic region belongs to the hg38 human genome; The dual liver cancer molecular marker detection reagent is a nucleic acid combination A for detecting the CpG methylation level of the chr2:25216400-25216752 genomic region and a nucleic acid combination B for detecting the CpG methylation level of the chr6:26250469-26250942 genomic region; the nucleic acid combination A includes a first primer pair combination and a first probe; The base sequence of the first primer pair combination and the base sequence of the first probe are shown in SEQ ID NO.1-3, SEQ ID NO.4-6, or SEQ ID NO.7-9; The nucleic acid combination B includes a second primer pair combination and a second probe; The base sequence of the second primer pair combination and the base sequence of the second probe are shown in SEQ ID NO.10-12, SEQ ID NO.13-15, SEQ ID NO.16-18, or SEQ ID NO.19-21; The sample detected by the dual liver cancer molecular marker detection reagent includes any one of an ex vivo tissue sample, an ex vivo plasma sample, an ex vivo serum sample, an ex vivo whole blood sample, an ex vivo blood cell sample, and a urine sample; The AUC value of the dual liver cancer molecular marker for methylation sequencing detection of liver cancer ex vivo blood samples was 0.951, the specificity was 0.901, and the sensitivity was 0.

917.

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

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