Tumor markers, kits and methods of use thereof for the diagnosis of early hepatocellular carcinoma

Abstract

The embodiments of the present application disclose a tumor marker, a kit and a use method for judging early hepatocellular carcinoma. The kit of the embodiments of the present application takes the first DNA shown as SEQ ID NO. 1 and / or the second DNA shown as SEQ ID NO. 2 as a judgment marker, judges whether the ex vivo sample is a positive sample of early hepatocellular carcinoma by judging the methylation degree of the CpG site in the first DNA and / or the second DNA in the ex vivo sample, thereby prompting whether the organism derived from the ex vivo sample has early hepatocellular carcinoma lesion. The kit of the embodiments of the present application has good specificity and sensitivity for the positive judgment of early hepatocellular carcinoma based on the specific tumor marker, and can be applied to the screening and judgment of early hepatocellular carcinoma.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to tumor markers, kits, and methods of use for identifying early-stage hepatocellular carcinoma. Background Technology

[0002] Liver cancer, especially hepatocellular carcinoma, is one of the most common malignant tumors in China. Most patients with hepatocellular carcinoma are diagnosed at an advanced stage, with a median survival of only 1 to 1.5 years. In contrast, the 5-year postoperative survival rate for early-stage hepatocellular carcinoma exceeds 70%. Therefore, early screening, early diagnosis, and early treatment of hepatocellular carcinoma can greatly improve the prognosis of patients.

[0003] Currently, alpha-fetoprotein (AFP) is commonly used as a blood biomarker for the early detection of hepatocellular carcinoma. However, AFP is easily affected by many factors, such as enzymes in the blood, leading to insufficient sensitivity and specificity when used as a blood biomarker for early detection of hepatocellular carcinoma. Summary of the Invention

[0004] This application provides a tumor marker, kit, and method of use for identifying early-stage hepatocellular carcinoma, which can solve the problem of insufficient sensitivity and specificity when using alpha-fetoprotein to identify early-stage hepatocellular carcinoma.

[0005] This application provides an example of using isolated tumor markers or combinations of tumor markers as tumor markers for early-stage hepatocellular carcinoma. The tumor markers include: a first DNA and / or a second DNA. The nucleotide sequence of the first DNA is shown in SEQ ID NO.1, and the nucleotide sequence of the second DNA is shown in SEQ ID NO.2. The first DNA and the second DNA include not only the base sequence information of the DNA but also naturally occurring DNA fragments. Each of the first DNA and the second DNA independently includes methylated CpG sites and / or unmethylated CpG sites.

[0006] Optionally, in some embodiments, all CpG sites of the first and / or second DNA used as tumor markers are methylated CpG sites. In other embodiments, all CpG sites of the first and / or second DNA used as tumor markers are unmethylated CpG sites.

[0007] This application provides an embodiment of an isolated tumor marker or combination of tumor markers as the determining factor in a kit for determining whether an ex vivo sample is a positive sample for early hepatocellular carcinoma. The tumor marker combination includes: a first DNA and / or a second DNA. The nucleotide sequence of the first DNA is shown in SEQ ID NO.1, and the nucleotide sequence of the second DNA is shown in SEQ ID NO.2. The first DNA and the second DNA each independently include methylated CpG sites and unmethylated CpG sites.

[0008] Optionally, in some embodiments, all CpG sites of the first and / or second DNA used as tumor markers are methylated CpG sites. In other embodiments, all CpG sites of the first and / or second DNA used as tumor markers are unmethylated CpG sites.

[0009] Optionally, in some embodiments, the first DNA is selected from the BNC1 gene at genomic coordinates chr15:83284254-83284648. The second DNA is selected from the DEUP1 gene at genomic coordinates chr11:93330461-93330772.

[0010] This application provides a kit for identifying positive samples of early-stage hepatocellular carcinoma. The kit identifies early-stage hepatocellular carcinoma by determining whether an ex vivo sample is positive. The kit includes a transformation reagent, a first amplification primer pair, and a first fluorescent label. In other embodiments, the kit may further include a second amplification primer pair and a second fluorescent label. In other embodiments, the kit may further include PCR amplification reagents.

[0011] In some embodiments, the conversion reagent can convert unmethylated cytosine in the first DNA containing methylated CpG sites in the cfDNA sample into uracil, obtaining a first transformed DNA strand A; the conversion reagent can also convert all cytosine in the first DNA not containing methylated CpG sites in the cfDNA sample into uracil, obtaining a first transformed DNA strand B. The nucleotide sequence of the first DNA is shown in SEQ ID NO.1.

[0012] In some embodiments, the conversion reagent can also convert unmethylated cytosine in the second DNA containing methylated CpG sites in the cfDNA sample into uracil to obtain the second transformed DNA strand A, and convert all cytosine in the second DNA without methylated CpG sites in the cfDNA sample into uracil to obtain the second transformed DNA strand B. The second DNA is shown in SEQ ID NO. 2. In some embodiments, the conversion reagent includes bisulfite.

[0013] In some embodiments, the first amplification primer pair is used for PCR amplification of the first transformed DNA strand A. The first fluorescent label includes a first luminescent molecule, a first oligonucleotide, and a first quencher molecule connected in sequence. After the first DNA containing methylated CpG sites is transformed into the first transformed DNA strand A, unmethylated cytosine is converted to uracil, but methylated cytosine at the CpG sites remains unchanged; after the first DNA not containing methylated CpG sites is transformed into the first transformed DNA strand B, all cytosine is converted to uracil. Therefore, the nucleotide sequences of the first transformed DNA strand A and the first transformed DNA strand B are different. The first amplification primer pair and the first fluorescent label in the embodiments of this application are designed specifically for the first transformed DNA strand A, and therefore can specifically amplify and bind to the first transformed DNA strand A, but will not amplify or bind to the first transformed DNA strand B.

[0014] In some embodiments, the second amplification primer pair is used for PCR amplification of the second transformed DNA molecule. The second fluorescent label includes a second luminescent molecule, a second oligonucleotide, and a second quencher molecule linked together in sequence. After the second DNA containing methylated CpG sites is converted into the second transformed DNA strand A, unmethylated cytosine is converted into uracil, but the methylated cytosine at the CpG sites remains unchanged; after the second DNA not containing methylated CpG sites is converted into the second transformed DNA strand B, all cytosine is converted into uracil. Therefore, the nucleotide sequences of the second transformed DNA strand A and the second transformed DNA strand B are different. The second amplification primer pair and the second fluorescent label in the embodiments of this application are designed specifically for the second transformed DNA strand A, thus they can specifically amplify and bind to the second transformed DNA strand A, but will not amplify or bind to the second transformed DNA strand B.

[0015] In some embodiments, during each cycle of PCR amplification of the cfDNA sample, the first oligonucleotide is integrated into each newly amplified first transformed DNA strand A, and the first luminescent molecule separates from the first quencher molecule. Once the first luminescent molecule separates from the first quencher molecule, its fluorescence emission is not affected by the quenching effect of the first quencher molecule. However, the first oligonucleotide does not integrate into the first transformed DNA strand B.

[0016] In some embodiments, during each cycle of PCR amplification of the cfDNA sample, the second oligonucleotide is integrated into each newly amplified strand A of the second transformed DNA, and the second luminescent molecule separates from the second quencher molecule. Once separated, the fluorescence emission of the second luminescent molecule is unaffected by the quenching effect of the second quencher molecule. However, the second oligonucleotide does not integrate into the strand B of the second transformed DNA.

[0017] In some embodiments, the first luminescent molecule and the second luminescent molecule are each independently selected from FAM, Cy5, VIC, TET, HEX, JOE, and ROX.

[0018] In some embodiments, the first luminescent molecule and the second luminescent molecule are different types of luminescent molecules.

[0019] In some embodiments, the cfDNA sample is derived from blood, plasma, or serum.

[0020] In some embodiments, PCR is quantitative real-time PCR, such as methylation-specific quantitative real-time PCR.

[0021] In some embodiments, the first amplification primer pair includes a first upstream primer and a first downstream primer. The nucleotide sequence of the first upstream primer is shown in SEQ ID NO.3, and the nucleotide sequence of the first downstream primer is shown in SEQ ID NO.4.

[0022] In some embodiments, the second amplification primer pair includes a second upstream primer and a second downstream primer. The nucleotide sequence of the second upstream primer is shown in SEQ ID NO.5, and the nucleotide sequence of the second downstream primer is shown in SEQ ID NO.6.

[0023] In some embodiments, the nucleotide sequence of the first oligonucleotide is shown in SEQ ID NO.7.

[0024] In some embodiments, the nucleotide sequence of the second oligonucleotide is shown in SEQ ID NO.8.

[0025] In some embodiments, the kit further includes a third amplification primer pair and a third fluorescent label. The third amplification primer pair is used for PCR amplification of a reference gene in a cfDNA sample. The third fluorescent label comprises a third luminescent molecule, a third oligonucleotide, and a third quencher molecule linked together in sequence. The third oligonucleotide has a partially identical or complementary sequence to the reference gene.

[0026] In some embodiments, the reference gene is selected from any one of the following: ACTB gene (β-actin gene, or actin gene), RNasn P gene (ribonuclease P gene), GAPDH gene (glyceraldehyde-3-phosphate dehydrogenase gene), TBP gene (TATA-binding protein gene), and HPRT gene (hypoxanthine phosphoribosyltransferase gene).

[0027] In some embodiments, the third amplification primer pair includes a third upstream primer and a third downstream primer. The nucleotide sequence of the third upstream primer is shown in SEQ ID NO. 9, and the nucleotide sequence of the third downstream primer is shown in SEQ ID NO. 10.

[0028] In some embodiments, the nucleotide sequence of the third oligonucleotide is shown in SEQ ID NO.11.

[0029] In some embodiments, during each cycle of PCR amplification of the cfDNA sample, a third oligonucleotide is integrated into each newly amplified reference gene in a one-to-one correspondence, and the third luminescent molecule is separated from the third quencher molecule.

[0030] In some embodiments, the third luminescent molecule is selected from FAM, Cy5, VIC, TET, HEX, JOE, and ROX.

[0031] In some embodiments, the third luminescent molecule is a different type of luminescent molecule from the first and second luminescent molecule, in order to avoid mutual interference of their respective fluorescence when the three luminescent molecules coexist in the PCR reaction solution.

[0032] In some embodiments, PCR is quantitative real-time PCR.

[0033] In some embodiments, the kit may further include extraction reagents and PCR amplification reagents, etc.

[0034] The extraction reagent is used to extract cfDNA samples from ex vivo samples. Ex vivo samples can be plasma, serum, or blood.

[0035] The PCR amplification reagent is used to perform PCR amplification of the first transformed DNA strand A using the first amplification primer pair and the first fluorescent label, and to perform PCR amplification of the second transformed DNA strand A using the second amplification primer pair and the second fluorescent label. However, the PCR amplification reagent does not perform PCR amplification of the first transformed DNA strand B, nor does it perform PCR amplification of the second transformed DNA strand B. When the PCR reaction solution also contains a third amplification primer pair and a third fluorescent label, the PCR amplification reagent can also perform PCR amplification of a reference gene using the third amplification primer pair and the third fluorescent label.

[0036] In some embodiments, the method for determining whether an ex vivo sample is a positive sample for early-stage hepatocellular carcinoma using the kit of this application is as follows:

[0037] The cfDNA sample was transformed using a transformation reagent. Then, the reagents were mixed to prepare a PCR reaction solution, which was then used for PCR amplification. The fluorescence intensity of the PCR reaction solution was monitored in real time. The PCR reaction solution included at least the transformed DNA sample, a first amplification primer pair, a first fluorescent label, a second amplification primer pair, a second fluorescent label, and PCR amplification reagents.

[0038] If the fluorescence intensity of the first luminescent molecule in the PCR reaction solution reaches or exceeds the first fluorescence threshold and the amplification cycle number (Ct value) of the first transformed DNA methyl strand is greater than the first cycle threshold (e.g., 38 to 44), the first DNA is determined to be methylation negative; otherwise, it is determined to be methylation positive.

[0039] Under the condition that the fluorescence intensity of the second luminescent molecule in the PCR reaction solution reaches or exceeds the second fluorescence threshold and the amplification cycle number (Ct value) of the second transformed DNA methyl strand is greater than the second cycle threshold (e.g., 38 to 44), the second transformed DNA molecule is judged to be methylation negative; otherwise, it is judged to be methylation positive.

[0040] If either or both of the first and second DNA samples are found to be methylated positive, the cfDNA sample is considered a positive sample for early-stage hepatocellular carcinoma; otherwise, the cfDNA sample is considered a negative sample for early-stage hepatocellular carcinoma. Specifically, this includes the following situations:

[0041] 1. If the first DNA in the cfDNA sample is determined to be methylation-negative, and the second DNA in the cfDNA sample is determined to be methylation-negative, then the cfDNA sample is determined to be a negative sample for early hepatocellular carcinoma.

[0042] 2. If the first DNA molecule in the cfDNA sample is determined to be methylated positive, and the second transformed DNA molecule in the cfDNA sample is determined to be methylated negative, then the cfDNA sample is determined to be a positive sample for early hepatocellular carcinoma.

[0043] 3. If the first DNA molecule in the cfDNA sample is determined to be methylation-negative, and the second transformed DNA molecule in the cfDNA sample is determined to be methylation-positive, then the cfDNA sample is determined to be a positive sample for early hepatocellular carcinoma.

[0044] 4. If the first DNA molecule in the cfDNA sample is determined to be methylated positive, and the second transformed DNA molecule in the cfDNA sample is determined to be methylated positive, then the cfDNA sample is determined to be a positive sample for early hepatocellular carcinoma.

[0045] In other embodiments, the PCR reaction solution further includes a third amplification primer pair and a third fluorescent label. In this case, if the fluorescence intensity of the third luminescent molecule in the PCR reaction solution reaches or exceeds the third fluorescence threshold and the amplification cycle number (Ct value) of the reference gene is less than or equal to the third cycle threshold (e.g., 30 to 32), the current PCR amplification is considered valid, and the result is reliable. Otherwise, it is considered invalid, and the result is unreliable, requiring repeated PCR amplification.

[0046] Some embodiments of this application provide a method for using the above-described reagent kit, which includes the following steps:

[0047] 1. Using a transformation reagent, unmethylated cytosine in the first DNA containing methylated CpG sites in the cfDNA sample is converted to uracil to obtain the first transformed DNA strand A. Then, all cytosine in the first DNA without methylated CpG sites in the cfDNA sample is converted to uracil to obtain the first transformed DNA strand B, thereby obtaining the first transformed sample. The nucleotide sequence of the first DNA is shown in SEQ ID NO.1.

[0048] 2. Provide a first amplification primer pair and a first fluorescent label; the first amplification primer pair is used for PCR amplification of the first transformed DNA strand A, but not for PCR amplification of the first transformed DNA strand B; the first fluorescent label includes a first luminescent molecule, a first oligonucleotide, and a first quencher molecule connected in sequence;

[0049] 3. Add the first amplification primer pair, the first fluorescent label, and PCR amplification reagent to the first transformed sample to obtain the first PCR reaction solution, and perform PCR amplification;

[0050] 4. Monitor the fluorescence intensity of the first PCR reaction solution. Once the fluorescence intensity of the first luminescent molecule in the first PCR reaction solution reaches or exceeds the first fluorescence threshold, record the required number of PCR amplification cycles.

[0051] 5. If the number of amplification cycles of the DNA methyl strand after the first transformation is greater than the first cycle threshold, the first DNA is determined to be methylation negative; otherwise, it is determined to be methylation positive.

[0052] If the first DNA sample is methylated positive, the cfDNA sample is considered a positive sample for early-stage hepatocellular carcinoma; otherwise, the cfDNA sample is considered a negative sample for early-stage hepatocellular carcinoma.

[0053] Some embodiments of this application provide a method of using the above-described kit, which includes treating an in vitro sample with an extraction reagent to obtain a cfDNA sample.

[0054] The method of using the above-described reagent kit provided in some embodiments of this application also includes:

[0055] Using a transformation reagent, unmethylated cytosine in the first DNA strand containing methylated CpG sites in the cfDNA sample was converted to uracil, yielding the first transformed DNA strand A. All cytosine in the first DNA strand without methylated CpG sites in the cfDNA sample was converted to uracil, yielding the first transformed DNA strand B, thus obtaining the first transformed sample. The nucleotide sequence of the first DNA is shown in SEQ ID NO.1. Using the same transformation reagent, unmethylated cytosine in the second DNA strand containing methylated CpG sites in the cfDNA sample was converted to uracil, yielding the second transformed DNA strand A. All cytosine in the second DNA strand without methylated CpG sites in the cfDNA sample was converted to uracil, yielding the second transformed DNA strand B, thus obtaining the second transformed sample. The nucleotide sequence of the second DNA is shown in SEQ ID NO.2.

[0056] 3. Provide a first amplification primer pair, a first fluorescent label, a second amplification primer pair, and a second fluorescent label. The first amplification primer pair is used for PCR amplification of the first transformed DNA strand A. The first fluorescent label comprises a first luminescent molecule, a first oligonucleotide, and a first quencher molecule linked together in sequence. The second amplification primer pair is used for PCR amplification of the second transformed DNA strand A. The second fluorescent label comprises a second luminescent molecule, a second oligonucleotide, and a second quencher molecule linked together in sequence.

[0057] 4. Add the first amplification primer pair, the first fluorescent label, the second amplification primer pair, the second fluorescent label, and PCR amplification reagent to the transformed DNA sample (containing the first and second transformed samples mentioned above) to obtain the second PCR reaction solution, and perform PCR amplification.

[0058] 5. Monitor the fluorescence intensity of the second PCR reaction solution. Once the fluorescence intensity of the first luminescent molecule in the second PCR reaction solution reaches or exceeds the first fluorescence threshold, and the fluorescence intensity of the second luminescent molecule reaches or exceeds the second fluorescence threshold, record the required number of PCR amplification cycles.

[0059] 6. If the number of amplification cycles of the DNA methyl chain after the first transformation is greater than the first cycle threshold, the first DNA is determined to be methylation negative; otherwise, it is determined to be methylation positive. If the number of amplification cycles of the DNA methyl chain after the second transformation is greater than the second cycle threshold, the second DNA is determined to be methylation negative; otherwise, it is determined to be methylation positive.

[0060] If either or both of the first and second DNA samples are found to be methylated positive, the cfDNA sample is considered a positive sample for early-stage hepatocellular carcinoma; otherwise, the cfDNA sample is considered a negative sample for early-stage hepatocellular carcinoma. Specific determination methods are detailed in the kit description section and will not be repeated here.

[0061] In some embodiments, the first cycle threshold is 38 to 44.

[0062] In some embodiments, the second cycle threshold is 38 to 44.

[0063] In some embodiments, the method of using the kit further includes the following steps:

[0064] 1. Provide a third amplification primer pair and a third fluorescent label; the third amplification primer pair is used for PCR amplification of the reference gene in the cfDNA sample; the third fluorescent label includes a third luminescent molecule, a third oligonucleotide, and a third quencher molecule linked together in sequence;

[0065] 2. Add the third amplification primer pair and the third fluorescent label to the first or second PCR reaction solution described above to perform PCR amplification;

[0066] 3. After the fluorescence intensity of the third luminescent molecule reaches or exceeds the third fluorescence threshold, record the required number of PCR amplification cycles;

[0067] 4. If the number of amplification cycles for the reference gene is less than or equal to the threshold of the third cycle, the PCR amplification is considered valid; otherwise, it is considered invalid.

[0068] In some embodiments of this application, the kit includes reagents capable of specifically determining the methylation level of DNA molecules as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0069] In some embodiments of this application, the kit also includes a positive control. All CpG sites in the nucleotide sequence of the aforementioned tumor markers are methylated to form the positive control; therefore, all CpG sites in the positive control are methylated CpG sites.

[0070] In some embodiments of this application, the kit also includes a negative control. All CpG sites in the nucleotide sequence of the tumor markers described above are demethylated or unmethylated to form the negative control. Therefore, all CpG sites in the negative control are unmethylated CpG sites, and it does not include any methylated CpG sites.

[0071] The tumor markers provided in this application can be used as positive and negative controls for diagnosing early-stage hepatocellular carcinoma, effectively indicating the diagnosis results and correcting their accuracy.

[0072] By employing the technical solutions in any of the above embodiments, this application has achieved the following beneficial effects.

[0073] The kit of this application uses a first DNA with a nucleotide sequence as shown in SEQ ID NO.1 and a second DNA with a nucleotide sequence as shown in SEQ ID NO.2 as diagnostic markers. This application utilizes a transformation reagent to convert the first DNA containing methylated CpG sites in the cfDNA sample into a first transformed DNA methyl chain, and / or to convert the second DNA containing methylated CpG sites in the cfDNA sample into a second transformed DNA methyl chain. The degree of methylation of CpG sites in the first DNA and / or the second DNA is determined by quantitative real-time PCR, thereby determining whether the cfDNA sample is a positive sample for early hepatocellular carcinoma, thus indicating whether the organism originating from the in vitro sample has developed early hepatocellular carcinoma lesions. Based on its specific tumor markers, the kit of this application has good specificity and sensitivity for the positive determination of early hepatocellular carcinoma and can be applied to the screening and diagnosis of early hepatocellular carcinoma. Detailed Implementation

[0074] The technical solutions in the embodiments of this application are described clearly and completely below. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0075] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of embodiments. Furthermore, in the description of this application, the term "comprising" means "including but not limited to". Various embodiments of the present invention may exist in a range format; it should be understood that the description in a range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the present invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values ​​within that range.

[0076] This application provides an example of the use of isolated tumor markers or combinations of tumor markers as tumor markers for early-stage hepatocellular carcinoma. The tumor markers include: a first DNA and / or a second DNA. The nucleotide sequence of the first DNA is shown in SEQ ID NO.1, and the nucleotide sequence of the second DNA is shown in SEQ ID NO.2. Each of the first and second DNA independently includes methylated CpG sites and unmethylated CpG sites.

[0077] In some embodiments, all CpG sites in the first and / or second DNA used for tumor markers are methylated CpG sites. In other embodiments, all CpG sites in the first and / or second DNA used for tumor markers are unmethylated CpG sites.

[0078] Experiments have shown that the degree of methylation at the CpG sites of the first and second DNA molecule in a subject's cfDNA sample can be used to determine whether the subject is a positive or negative patient for early-stage hepatocellular carcinoma. If the subject is determined to be a positive patient for early-stage hepatocellular carcinoma, further assessment can be made based on histological examination, imaging findings, or other physicochemical indicators.

[0079] The first DNA molecule corresponds to the BNC1 gene, with genomic coordinates chr15:83284254-83284648. The second DNA molecule corresponds to the DEUP1 gene, with genomic coordinates chr11:93330461-93330772. Both genomic coordinates are from the hg38 version of the human reference genome.

[0080] In some embodiments of this application, "isolated" tumor markers refer to tumor markers that have been extracted, isolated, and purified from an individual or biological sample, or tumor markers synthesized by chemical / biological methods known in the art, which only need to have the above-mentioned nucleotide sequence.

[0081] In embodiments of this application, tumor markers can be artificially synthesized into nucleotide sequences with all CpG sites methylated, thereby serving as positive controls. In other embodiments, tumor marker combinations can be artificially synthesized into nucleotide sequences including all methylated CpG sites, thereby serving as positive controls.

[0082] In the embodiments of this application, "methylation" is a form of DNA chemical modification that can alter genetic expression without changing the DNA sequence. DNA methylation refers to the covalent binding of a methyl group to the 5th carbon position of cytosine in a CpG dinucleotide of the genome under the action of DNA methyltransferases. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression. In mammals, DNA methylation generally occurs at the cytosine in the cytosine-phosphate-guanine (CpG) dinucleotide site, manifested as 5-methylcytosine (5mC), while cytosine in non-CpG sites is generally not methylated. After treating DNA with bisulfite, unmethylated cytosine in the DNA is converted to uracil, while methylated cytosine remains unchanged. Therefore, under normal circumstances, if a DNA molecule contains methylated CpG, then after bisulfite treatment, the cytosine in the methylated CpG remains unchanged due to methylation modification, while other unmethylated cytosines are converted into uracil; if the DNA molecule does not contain methylated CpG, then all cytosines will be converted into uracil because they are not methylated.

[0083] In the embodiments of this application, "nucleotide" or "nucleic acid" refers to a molecule having two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. Oligonucleotides can be produced by any means, including chemical synthesis, DNA replication, reverse transcription, or combinations thereof. Typical deoxyribonucleotides of DNA are thymine, adenine, cytosine, and guanine. Typical ribonucleotides of RNA are uracil, adenine, cytosine, and guanine.

[0084] This application provides the use of isolated tumor markers or combinations of tumor markers in the preparation of a kit for identifying early-stage hepatocellular carcinoma. The tumor markers include: a first DNA and / or a second DNA. The nucleotide sequence of the first DNA is shown in SEQ ID NO.1, and the nucleotide sequence of the second DNA is shown in SEQ ID NO.2.

[0085] In some embodiments, all CpG sites of the first and / or second DNA used as tumor markers are methylated CpG sites. Nucleotide sequences in which all CpG sites are methylated can be used as positive controls.

[0086] In some embodiments, all CpG sites of the first and / or second DNA used as tumor markers are unmethylated CpG sites. Nucleotide sequences where all CpG sites are unmethylated can serve as a negative reference.

[0087] This application provides an embodiment of an isolated tumor marker combination as the judgment object for a kit used to determine whether an ex vivo sample is a positive sample for early hepatocellular carcinoma. The tumor marker combination includes: a first DNA and a second DNA. The nucleotide sequence of the first DNA is shown in SEQ ID NO.1, and the nucleotide sequence of the second DNA is shown in SEQ ID NO.2. The first DNA and the second DNA each independently include methylated CpG sites and unmethylated CpG sites, or all CpG sites of the first DNA and / or the second DNA are methylated CpG sites, or all CpG sites of the first DNA and / or the second DNA are unmethylated CpG sites.

[0088] This application provides a method for using the above-mentioned reagent kit, which includes the following steps:

[0089] 1. Using a transformation reagent, unmethylated cytosine in the first DNA strand containing methylated CpG sites in the cfDNA sample is converted to uracil, yielding the first transformed DNA strand A; all cytosine in the first DNA strand without methylated CpG sites in the cfDNA sample is converted to uracil, yielding the first transformed DNA strand B, thus obtaining the first transformed sample. The nucleotide sequence of the first DNA is shown in SEQ ID NO.1. The transformation reagent can be the Zymo bisulfite transformation kit.

[0090] 2. Provide a first amplification primer pair and a first fluorescent label; the first amplification primer pair is used for PCR amplification of the first transformed DNA strand A, but not for PCR amplification of the first transformed DNA strand B. The first fluorescent label comprises a first luminescent molecule, a first oligonucleotide, and a first quencher molecule linked together in sequence.

[0091] 3. Add the first amplification primer pair, the first fluorescent label, and PCR amplification reagent to the first transformed sample to obtain the first PCR reaction solution, and perform PCR amplification.

[0092] 4. Monitor the fluorescence intensity of the first PCR reaction solution. Once the fluorescence intensity of the first luminescent molecule in the first PCR reaction solution reaches or exceeds the first fluorescence threshold, record the required number of PCR amplification cycles.

[0093] If the number of amplification cycles of the DNA methyl strand after the first transformation is greater than the first cycle threshold, the first DNA is determined to be methylation negative; otherwise, it is determined to be methylation positive.

[0094] If the first DNA sample is methylated positive, the cfDNA sample is considered a positive sample for early-stage hepatocellular carcinoma; otherwise, the cfDNA sample is considered a negative sample for early-stage hepatocellular carcinoma.

[0095] This application provides a kit for identifying early-stage hepatocellular carcinoma, used to determine whether an ex vivo sample is a positive sample for early-stage hepatocellular carcinoma. The kit includes: a first amplification primer pair, a first fluorescent label, a second amplification primer pair, and a second fluorescent label.

[0096] In this embodiment, the first amplification primer pair is used for PCR amplification of the first transformed DNA strand A, but not for PCR amplification of the first transformed DNA strand B. The PCR in this application can be methylation-specific quantitative PCR. In the exponential phase of PCR amplification, the Ct value (Cycle Threshold) and final fluorescence intensity of the amplified DNA have a linear relationship with the initial copy number of the DNA, thus enabling quantification.

[0097] The first fluorescent label comprises a first luminescent molecule, a first oligonucleotide, and a first quencher molecule, linked sequentially. The first luminescent molecule can be selected from luminescent molecules such as FAM, Cy5, VIC, TET, HEX, JOE, and ROX. The properties of each of the above luminescent molecules are shown in Table 1 below.

[0098] Table 1 Properties of each luminescent molecule

[0099]

[0100] The first quenching molecule can be selected from BHQ-1 carboxylic acid, BHQ-2 carboxylic acid, BHQ-3 carboxylic acid, carboxytetramethylrhodamine (TAMRA), and 4-(4-oxaneaminophenylazo)benzoic acid (DABCYL). For example, the quenching molecule for VIC can be BHQ1, and the quenching molecule for Cy5 can be BHQ2.

[0101] In the first fluorescent label, the first luminescent molecule is attached to the 5' end of the first oligonucleotide, while the first quencher molecule is attached to the 3' end. When the first fluorescent label is intact, the fluorescent signal emitted by the first luminescent molecule is absorbed by the first quencher molecule, thus the first fluorescent label does not emit a fluorescent signal. During the exponential phase of methylation-specific quantitative PCR amplification, the 5'-3' exonuclease activity of Taq polymerase degrades the first fluorescent label, separating the first luminescent molecule from the first quencher molecule. Sequences in the first oligonucleotide that are identical or complementary to the first transformed DNA strand A are then integrated into each newly amplified first transformed DNA strand A. Therefore, when each first fluorescent label is degraded and some relevant sequences are integrated into each newly amplified first transformed DNA strand A, one first luminescent molecule is released. At this point, the first luminescent molecule separates from the first quencher molecule, and its fluorescence is not quenched by the first quencher molecule. Therefore, there is a linear relationship between the number of first luminescent molecules and the fluorescence intensity and the number of amplified first transformed DNA strands A, thus achieving quantification. The first oligonucleotide has a partially identical or complementary sequence to the first transformed DNA strand A. Therefore, during the exponential phase of PCR amplification, i.e., in each cycle of PCR amplification of the DNA sample, the identical or complementary sequences in the first oligonucleotide to the first transformed DNA strand A are integrated one-to-one into each newly amplified first transformed DNA strand A. In the embodiments of this application, the first amplification primer pair and the first fluorescent label are designed specifically for the first transformed DNA strand A, thus enabling them to specifically amplify and bind to the first transformed DNA strand A, while not amplifying or binding to the first transformed DNA strand B.

[0102] The second amplification primer pair is used for PCR amplification of the second transformed DNA strand A in the transformed cfDNA sample, but not for PCR amplification of the second transformed DNA strand B.

[0103] The second fluorescent label comprises a second luminescent molecule, a second oligonucleotide, and a second quencher molecule linked together in sequence. The second luminescent molecule can be selected from luminescent molecules such as FAM, Cy5, VIC, TET, HEX, JOE, and ROX, but it must be a different type of luminescent molecule from the first luminescent molecule to avoid interference between the fluorescence emitted by different luminescent molecules. The second quencher molecule can be selected from BHQ-1 carboxylic acid, BHQ-2 carboxylic acid, BHQ-3 carboxylic acid, carboxytetramethylrhodamine (TAMRA), and 4-(4-oxaneaminophenylazo)benzoic acid (DABCYL). In the second fluorescent label, the second luminescent molecule is linked to the 5' end of the second oligonucleotide, while the second quencher molecule is linked to the 3' end of the second oligonucleotide. The mechanism of action of the second fluorescent label can be referenced from that of the first fluorescent label. The second oligonucleotide has a partially identical or complementary sequence to the second transformed DNA strand A. Therefore, in each cycle of PCR amplification of the transformed cfDNA sample, the identical or complementary sequences in the second oligonucleotide are integrated into each newly amplified second transformed DNA strand A. In the embodiments of this application, the second amplification primer pair and the second fluorescent label are designed specifically for the second transformed DNA strand A, thus enabling them to specifically amplify and bind to the second transformed DNA strand A, while not amplifying or binding to the second transformed DNA strand B.

[0104] In embodiments of this application, the DNA sample is derived from blood, plasma, or serum. For example, the DNA sample may be cell-free DNA (cfDNA). cfDNA is fragmented DNA found in human peripheral blood and other circulating body fluids.

[0105] cfDNA in the blood can primarily exist in the form of wrapped nucleosomes, because cfDNA not wrapped around nucleosomes in this form is rapidly degraded. A single nucleosome contains approximately 170 bp of cfDNA; therefore, the major fragment of cfDNA exhibits a main peak at 170 bp (equivalent to one nucleosome), followed by a smaller peak at 340 bp (equivalent to two nucleosomes), and so on.

[0106] In some other embodiments, the length of cfDNA may also be between 170bp and 210bp (inclusive). However, in other embodiments, the length of cfDNA may also be 180bp, 185bp, 190bp, 195bp, 200bp, etc.

[0107] In other embodiments, the length of the cfDNA can be an integer multiple of any value between 170 bp and 210 bp (determined by the number of nucleosomes wrapped around it). For example, 340 bp, 420 bp, etc.

[0108] Studies have shown that cfDNA in the plasma of cancer patients can carry mutations and epigenetic aberrations in tumor DNA, especially abnormal DNA methylation changes in the early stages of hepatocellular carcinoma (HCC). In mammals, DNA methylation mainly occurs on cytosine in cytosine-phosphate-guanine (CpG) dinucleotides, manifesting as 5-methylcytosine (5mC). Compared to normal liver tissue, HCC tissue often exhibits global hypomethylation of genomic DNA, as well as local or global hypermethylation of DNA located on CpG islands. Abnormal hypermethylation of CpG islands can regulate the expression of cancer-related genes, thus playing an important role in the occurrence and development of HCC. The inventors' research found that abnormal methylation levels of the BNC1 and DEUP1 genes are closely related to early-stage HCC. This is manifested in the plasma of patients with early-stage HCC, where related sequences of the BNC1 and DEUP1 genes are abnormally methylated, while in the plasma of individuals without HCC, these sequences are not methylated. Therefore, this application embodiment determines early hepatocellular carcinoma by judging the methylation level of specific sequences of the BNC1 and DEUP1 genes in the cfDNA sample.

[0109] In embodiments of this application, the first amplification primer pair includes a first upstream primer and a first downstream primer. The nucleotide sequence of the first upstream primer is shown in SEQ ID NO.3, and the nucleotide sequence of the first downstream primer is shown in SEQ ID NO.4. The second amplification primer pair includes a second upstream primer and a second downstream primer. The nucleotide sequence of the second upstream primer is shown in SEQ ID NO.5, and the nucleotide sequence of the second downstream primer is shown in SEQ ID NO.6.

[0110] In embodiments of this application, the nucleotide sequence of the first oligonucleotide is shown in SEQ ID NO. 7. This nucleotide sequence is capable of embedding into each newly amplified first transformed DNA strand A in each cycle of PCR amplification, and the first luminescent molecule and the first quencher molecule also separate as the structural integrity of the first fluorescent label is disrupted. The first oligonucleotide does not embed into the first transformed DNA strand B.

[0111] In embodiments of this application, the nucleotide sequence of the second oligonucleotide is shown in SEQ ID NO. 8. This nucleotide sequence is capable of embedding into each newly amplified second transformed DNA strand A in each cycle of PCR amplification, and the second luminescent molecule and the second quencher molecule also separate as the structural integrity of the second fluorescent label is disrupted. The second oligonucleotide does not embed into the second transformed DNA strand B.

[0112] In embodiments of this application, the kit further includes: a third amplification primer pair and a third fluorescent label.

[0113] The third amplification primer pair is used for PCR amplification of the reference gene in the cfDNA sample. The reference gene is selected from any one of the following: ACTB gene (β-actin gene, or actin gene), RNaSN P gene (ribonuclease P gene), GAPDH gene (glyceraldehyde-3-phosphate dehydrogenase gene), TBP gene (TATA-binding protein gene), and HPRT gene (hypoxanthine phosphoribosyltransferase gene). The third amplification primer pair includes a third upstream primer and a third downstream primer. The nucleotide sequence of the third upstream primer is shown in SEQ ID NO.9, and the nucleotide sequence of the third downstream primer is shown in SEQ ID NO.10. The genomic coordinates of the ACTB gene-related region are chr7:5529432-5529682, and its nucleotide sequence is shown in SEQ ID NO.12.

[0114] The third fluorescent label comprises a third luminescent molecule, a third oligonucleotide, and a third quencher molecule linked together. The third luminescent molecule can be selected from luminescent molecules such as FAM, Cy5, VIC, TET, HEX, JOE, and ROX, but it must be a different type of luminescent molecule from the first and second luminescent molecules to prevent fluorescence interference. The third quencher molecule can be selected from BHQ-1 carboxylic acid, BHQ-2 carboxylic acid, BHQ-3 carboxylic acid, carboxytetramethylrhodamine (TAMRA), and 4-(4-oxaneaminophenylazo)benzoic acid (DABCYL). The mechanism of action of the third luminescent molecule and the third quencher molecule is the same as that of the first luminescent molecule and the first quencher molecule. The nucleotide sequence of the third oligonucleotide is shown in SEQ ID NO. 11. This base sequence can be inserted into each newly amplified reference gene in each cycle of PCR amplification, and the third luminescent molecule and the third quencher molecule will separate as the structural integrity of the third fluorescent label is disrupted.

[0115] In some embodiments, the kit may further include extraction reagents and / or PCR amplification reagents, etc.

[0116] The extraction reagent is used to extract cfDNA from ex vivo samples. Ex vivo samples can be plasma, serum, or blood. The QIAamp Circulating Nucleic Acid Kit (Qiagen) was used as the extraction reagent to extract cfDNA from serum samples.

[0117] The PCR amplification system consisted of 25 μL of Taq Pro HS Probe Master Mix. The amount of transformed cfDNA sample added was 10 μL. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 120 s; followed by 50 cycles at 95°C for 10 s and then at 60°C for 30 s; finally, storage at 4°C. When a third primer pair and a third fluorescent label are present in the PCR reaction solution, the PCR amplification reagent can also amplify the reference gene in the cfDNA sample using these primer pairs and fluorescent labels.

[0118] In some embodiments, this application also provides a method for using the reagent kit, which includes the following steps:

[0119] 1.1 Using a transformation reagent, unmethylated cytosine in the first DNA layer containing methylated CpG sites in the cfDNA sample was converted to uracil to obtain the first transformed DNA strand A. All cytosine in the first DNA layer without methylated CpG sites in the cfDNA sample was converted to uracil to obtain the first transformed DNA strand B, thus obtaining the first transformed sample. Similarly, using a transformation reagent, unmethylated cytosine in the second DNA layer containing methylated CpG sites in the cfDNA sample was converted to uracil to obtain the second transformed DNA strand A. All cytosine in the second DNA layer without methylated CpG sites in the cfDNA sample was converted to uracil to obtain the second transformed DNA strand B, thus obtaining the second transformed sample.

[0120] 1.2 Provides a first amplification primer pair, a first fluorescent label, a second amplification primer pair, and a second fluorescent label; the first amplification primer pair is used for PCR amplification of the first transformed DNA strand A, but not for PCR amplification of the first transformed DNA strand B; the first fluorescent label comprises a first luminescent molecule, a first oligonucleotide, and a first quencher molecule connected in sequence; the second amplification primer pair is used for PCR amplification of the second transformed DNA strand A, but not for PCR amplification of the second transformed DNA strand B; the second fluorescent label comprises a second luminescent molecule, a second oligonucleotide, and a second quencher molecule connected in sequence.

[0121] 1.3 Add the first amplification primer pair, the first fluorescent label, the second amplification primer pair, the second fluorescent label, and PCR amplification reagent to the transformed DNA samples (including the first and second transformed samples) to obtain the second PCR reaction solution, and perform PCR amplification. Monitor the fluorescence intensity of the second PCR reaction solution. After the fluorescence intensity of the first luminescent molecule in the second PCR reaction solution reaches or exceeds the first fluorescence threshold, and the fluorescence intensity of the second luminescent molecule reaches or exceeds the second fluorescence threshold, record the required number of PCR amplification cycles.

[0122] 1.4 If the amplification cycle number of the DNA methyl chain after the first transformation is greater than the first cycle threshold, the first DNA is determined to be methylation-negative; otherwise, it is determined to be methylation-positive. If the amplification cycle number of the DNA methyl chain after the second transformation is greater than the second cycle threshold, the second DNA is determined to be methylation-negative; otherwise, it is determined to be methylation-positive. If either the first or second DNA is determined to be methylation-positive, the cfDNA sample is determined to be a positive sample for early hepatocellular carcinoma; otherwise, the cfDNA sample is determined to be a negative sample for early hepatocellular carcinoma.

[0123] The threshold for the first cycle can be between 38 and 44, for example, 38, 39, 40, 41, 42, 43, and 44.

[0124] The threshold for the second cycle can be 38 to 44, for example, 38, 39, 40, 41, 42, 43, 44.

[0125] In some embodiments, the method of using the kit includes the following steps: treating the in vitro sample with an extraction reagent to obtain the above-mentioned cfDNA sample.

[0126] In some embodiments, the method of using the kit includes the following steps:

[0127] 3.1 Provide a third amplification primer pair and a third fluorescent label; the third amplification primer pair is used for PCR amplification of the reference gene in the DNA sample; the third fluorescent label includes a third luminescent molecule, a third oligonucleotide, and a third quencher molecule linked together in sequence;

[0128] 3.2 Add the third amplification primer pair and the third fluorescent label together with the PCR amplification reagent to the transformed cfDNA sample (e.g., the first or second transformed sample mentioned above) or to the first or second PCR reaction solution mentioned above, and perform PCR amplification; after the fluorescence intensity of the third luminescent molecule reaches or exceeds the third fluorescence threshold, record the required number of PCR amplification cycles.

[0129] 3.3 If the number of amplification cycles for the reference gene is less than or equal to the threshold of the third cycle, the PCR amplification is considered valid; otherwise, it is considered invalid.

[0130] Step 3.3 is used to determine whether a particular PCR amplification product is reliable. If it is reliable, the PCR amplification result is valid, and further evaluation can proceed. If it is not reliable, the PCR amplification result is invalid, and a new PCR amplification needs to be performed.

[0131] In the method of using the kit described in the above embodiments, it is necessary to obtain the number of cycles (i.e., Ct value) required for the fluorescence signal of each gene to reach the fluorescence threshold. Each gene mentioned above refers to the BNC1 gene corresponding to the first DNA, the DEUP1 gene corresponding to the second DNA, and the ACTB gene corresponding to the reference gene. However, the reference gene is not limited to the ACTB gene.

[0132] The present application will be further described below with reference to specific embodiments.

[0133] The ex vivo samples used in the various embodiments of this application include: blood samples from 100 healthy individuals (without hepatocellular carcinoma), blood samples from 150 patients with hepatitis B and cirrhosis (without hepatocellular carcinoma), and blood samples from 150 patients with hepatocellular carcinoma. All blood samples were stored in cell-free DNA-specific blood collection tubes (Streck), containing anticoagulants and cell stabilizers. The ex vivo samples were then processed using the kits described in the embodiments of this application according to the following methods.

[0134] The method in this embodiment includes the following steps:

[0135] 1. The ex vivo sample (i.e., blood sample) is treated with extraction reagents to obtain cfDNA samples. This step specifically includes:

[0136] The above-mentioned ex vivo samples (blood samples) were centrifuged to obtain plasma samples. cfDNA samples were extracted from the plasma samples using the QIAamp Circulating Nucleic Acid Kit (Qiagen). The extraction method was performed according to the kit's instruction manual.

[0137] 2. The cfDNA sample is treated with a transformation reagent to convert unmethylated cytosine in the cfDNA sample to uracil, resulting in a transformed cfDNA sample (containing a first transformed DNA A strand, a first transformed DNA B strand, a second transformed DNA A strand, and a second transformed DNA B strand). This step specifically includes:

[0138] Take 10 ng of cfDNA from the cfDNA sample obtained in step 1 and transform it using the Zymo bisulfite transformation kit. The transformation process converts all unmethylated cytosine in the cfDNA sample to uracil, while methylated cytosine remains unchanged.

[0139] 3. Provide the first amplification primer pair, the first fluorescent label, the second amplification primer pair, the second fluorescent label, the third amplification primer pair, the third fluorescent label, and PCR amplification reagents, as detailed below:

[0140] The first amplification primer pair includes a first upstream primer as shown in SEQ ID NO.3 and a first downstream primer as shown in SEQ ID NO.4. The first amplification primer pair is used for PCR amplification of the first transformed DNA strand A in the transformed cfDNA sample. The first DNA originates from the BNC1 gene.

[0141] The first fluorescent label comprises a first luminescent molecule, a first oligonucleotide, and a first quencher molecule, linked sequentially. The first luminescent molecule is FAM. The nucleotide sequence of the first oligonucleotide is shown in SEQ ID NO.7. The first quencher molecule is BHQ-3.

[0142] The second amplification primer pair includes a second upstream primer as shown in SEQ ID NO. 5 and a second downstream primer as shown in SEQ ID NO. 6. This second amplification primer pair is used for PCR amplification of the second transformed DNA strand A. The second DNA originates from the DEUP1 gene.

[0143] The second fluorescent label comprises a second luminescent molecule, a second oligonucleotide, and a second quencher molecule linked together in sequence. The second luminescent molecule is Cy5. The nucleotide sequence of the second oligonucleotide is shown in SEQ ID NO. 8. The second quencher molecule is BHQ-2.

[0144] The third amplification primer pair includes a third upstream primer as shown in SEQ ID NO. 9 and a third downstream primer as shown in SEQ ID NO. 10. The third amplification primer pair is used for PCR amplification of the reference gene, which is the ACTB gene, and its nucleotide sequence is shown in SEQ ID NO. 12.

[0145] The third fluorescent label comprises a third luminescent molecule, a third oligonucleotide, and a third quencher molecule linked together in sequence. The third luminescent molecule is VIC. The nucleotide sequence of the third oligonucleotide is shown in SEQ ID NO.11. The third quencher molecule is BHQ-1.

[0146] PCR amplification reagents include Taq Pro HS Probe Master Mix, which contains DNA polymerase, dNTPs, and buffer.

[0147] 4. Add the first amplification primer pair, the first fluorescent label, the second amplification primer pair, the second fluorescent label, the third amplification primer pair, the third fluorescent label, and PCR amplification reagent to the DNA sample to obtain the PCR reaction solution, and perform PCR amplification. Determine the fluorescence intensity of the obtained PCR reaction solution. After the fluorescence values ​​of the first, second, and third luminescent molecules all exceed their respective fluorescence thresholds, record the required number of PCR amplification cycles. Use a Bio-Rad quantitative PCR instrument for PCR amplification and fluorescence determination. Specifically, the fluorescence value of the first luminescent molecule must exceed the first fluorescence threshold. The fluorescence value of the second luminescent molecule must exceed the second fluorescence threshold. The fluorescence value of the third luminescent molecule must exceed the third fluorescence threshold.

[0148] The components of the PCR reaction solution are shown in Table 2 below.

[0149] Table 2 Components of PCR Reaction Solution

[0150] Components Reagent Specifications Volume (μL) Taq Pro HS Probe Master Mix 2× 25 Upstream primer (or forward primer) 10μM 1.5 Downstream primer (or reverse primer) 10μM 1.5 Fluorescent markers (or fluorescent probes) 10μM 1 DNA sample (transformed DNA) / 10 <![CDATA[ddH2O]]> / Add to 50

[0151] In Table 2, the upstream primers include a first upstream primer, a second upstream primer, and a third upstream primer. The downstream primers include a first downstream primer, a second downstream primer, and a third downstream primer. The fluorescent labels include a first fluorescent label, a second fluorescent label, and a third fluorescent label. In this embodiment, the above primers and fluorescent labels are mixed with the DNA sample for detection because the first luminescent molecule FAM emits green light, the second luminescent molecule Cy5 emits red light, and the third luminescent molecule VIC emits yellow light. The three luminescent molecules emit different colors and will not interfere with each other; therefore, they can be mixed together for detection. In other embodiments, the first amplification primer pair and the first fluorescent label, the second amplification primer pair and the second fluorescent label, and the third amplification primer pair and the third fluorescent label can also be mixed with the transformed cfDNA sample and PCR amplification reagent, respectively, and detected separately.

[0152] The conditions for the PCR reaction are shown in Table 3 below.

[0153] Table 3 Conditions for PCR reaction

[0154]

[0155] 5. If the amplification cycle number (Ct value) of the DNA methyl chain (corresponding to the BNC1 gene) after the first transformation is greater than the first cycle threshold (value is 40), the first DNA is judged as methylation negative; otherwise, it is judged as methylation positive. Because methylated cfDNA originates from cancer cells, the number of cancer cells determines the abundance and content of methylated cfDNA. The higher the degree of methylation in the cfDNA sample, the smaller the Ct value. Conversely, the lower the degree of methylation in the cfDNA sample, the larger the Ct value. Therefore, the degree of methylation can be determined based on the Ct value.

[0156] If the amplification cycle number (Ct value) of the DNA methyl chain (corresponding to the DEUP1 gene) after the second transformation is greater than the second cycle threshold (value of 40), the second DNA is determined to be methylation negative; otherwise, it is determined to be methylation positive. If either the first or second DNA is determined to be methylation positive, the DNA sample is determined to be a positive sample for early hepatocellular carcinoma; otherwise, the DNA sample is determined to be a negative sample for early hepatocellular carcinoma.

[0157] Specifically, this includes the following situations:

[0158] 1. If the first DNA in a DNA sample is determined to be methylation-negative, and the second DNA in the DNA sample is also determined to be methylation-negative, then the DNA sample is determined to be a negative sample for early-stage hepatocellular carcinoma.

[0159] 2. If the first DNA in the DNA sample is determined to be methylated positive and the second DNA in the DNA sample is determined to be methylated negative, then the DNA sample is determined to be a positive sample for early hepatocellular carcinoma.

[0160] 3. If the first DNA in the DNA sample is determined to be methylation-negative, and the second DNA in the DNA sample is determined to be methylation-positive, then the DNA sample is determined to be a positive sample for early hepatocellular carcinoma.

[0161] 4. If the first DNA in the DNA sample is determined to be methylated positive, and the second DNA in the DNA sample is determined to be methylated positive, then the DNA sample is determined to be a positive sample for early hepatocellular carcinoma.

[0162] Furthermore, if the fluorescence intensity of the third luminescent molecule in the PCR reaction solution reaches or exceeds the third fluorescence threshold, and the amplification cycle number (Ct value) of the reference gene is less than or equal to the third cycle threshold (e.g., 30 to 32), the PCR amplification is considered valid, and the result is reliable. Otherwise, it is considered invalid, and the result is unreliable, requiring repeated PCR amplification. After excluding unreliable results, the reliable results are statistically analyzed, and the results are shown in Table 4.

[0163] In addition, since alpha-fetoprotein (AFP) is a common blood marker for diagnosing hepatocellular carcinoma, the AFP results were used as a control. The AFP concentration in the ex vivo samples (blood samples) was determined using an AFP assay kit (immunochromatography). A serum AFP concentration greater than 20 ng / μL was considered a positive sample for hepatocellular carcinoma; a serum AFP concentration less than or equal to 20 ng / μL was considered a negative sample for hepatocellular carcinoma. The results were compared with the pathological examination results of the subjects to calculate the sensitivity and specificity of using AFP for diagnosis. The results are shown in Table 4.

[0164] Table 4. Judgment Results Table in the Embodiments of this Application

[0165]

[0166] As shown in Table 4, when only the BNC1 and DEUP1 genes are evaluated, their sensitivity and specificity are already higher than those of AFP. Furthermore, when both BNC1 and DEUP1 genes are evaluated together, a high level of sensitivity and specificity for hepatocellular carcinoma detection is achieved, significantly outperforming the traditional serum protein marker AFP.

[0167] Those skilled in the art will understand that the methylation level of the above-mentioned nucleotide sequence can be determined using methods known in the art for determining DNA methylation levels, such as methylation-specific PCR, bisulfite treatment and sequencing, restriction endonuclease analysis combined with sodium bisulfite, methylation-specific quantitative PCR, methylation-sensitive high-resolution melting curve analysis, or pyrosequencing. Accordingly, the reaction reagents and the determination reagents are necessary for the above methods. In some embodiments, the reaction reagents are selected from bisulfite or its derivatives.

[0168] In this application, "patient" or "subject" refers to a human being diagnosed with early-stage hepatocellular carcinoma for the first time. A patient may be an individual receiving treatment for early-stage hepatocellular carcinoma or any individual who wishes to use the methods described in this application for assessment or diagnosis.

[0169] "Methylation level" represents the proportion of one or more sites in a nucleotide sequence that are methylated. For example, the methylation level of a region / interval in whole-genome DNA refers to the proportion of all CpG sites in that region / interval that are methylated, while the average methylation level of whole-genome DNA refers to the mean of the methylation levels of all regions / intervals in whole-genome DNA. Those skilled in the art know the process of converting the results of methods for determining DNA methylation (e.g., simplified methylation sequencing) into methylation levels.

[0170] In some embodiments of this application, the DNA may be single-stranded DNA (ssDNA). In some embodiments of this application, the DNA may be double-stranded DNA (dsDNA).

[0171] In this application, the terms "DNA strand A" and "DNA strand B" are used only to distinguish different DNA molecules and do not imply that DNA strand A and DNA strand B are complementary strands of the same DNA molecule. For example, DNA strand A after the first transformation and DNA strand B after the first transformation are different DNA molecules with different nucleotide sequences, but they are not complementary. Similarly, DNA strand A after the second transformation and DNA strand B after the second transformation are different DNA molecules with different nucleotide sequences, but they are not complementary.

[0172] The preferred implementation methods and materials described herein are for illustrative purposes only and should not be used to limit the content of this application.

[0173] Unless otherwise stated, the raw materials and reagents used in the embodiments of this application are all commercially available products, or can be prepared by methods known in the art.

[0174] The kits used in this application embodiment, the QIAamp Circulating Nucleic Acid Kit (Qiagen), were purchased from Qiagen Biotechnology (Shanghai) Co., Ltd. The Zymo bisulfite conversion kit was purchased from Zymo Biotech. The AFP concentration in ex vivo samples (serum samples) was determined using an alpha-fetoprotein assay kit (immunochromatography). The PCR amplification kit was purchased from Novizan Biotech. The real-time PCR instrument was purchased from Bio-Rad.

[0175] All operations in the embodiments of this application conform to the manufacturer's instructions.

[0176] The above provides a detailed description of the tumor markers, reagent kits, and applications for identifying early-stage hepatocellular carcinoma provided in the embodiments of this application. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the embodiments above are only for the purpose of helping to understand the methods and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A kit for identifying positive samples of early hepatocellular carcinoma, characterized in that, The kit includes: The transformation reagent converts unmethylated cytosine in the first DNA containing methylated CpG sites in the cfDNA sample into uracil to obtain a first transformed DNA chain A. It then converts all cytosine in the first DNA of the cfDNA sample that does not contain methylated CpG sites into uracil to obtain a first transformed DNA chain B. The nucleotide sequence of the first DNA is shown in SEQ ID NO.

1. The reagent also converts unmethylated cytosine in the second DNA containing methylated CpG sites in the cfDNA sample into uracil to obtain a second transformed DNA chain A. Finally, it converts all cytosine in the second DNA of the cfDNA sample that does not contain methylated CpG sites into uracil to obtain a second transformed DNA chain B. The nucleotide sequence of the second DNA is shown in SEQ ID NO.

2. A first amplification primer pair is used for PCR amplification of the first transformed DNA strand A, and a first fluorescent label comprising a first luminescent molecule, a first oligonucleotide, and a first quencher molecule connected in sequence; and a second amplification primer pair is used for PCR amplification of the second transformed DNA strand A, and a second fluorescent label comprising a second luminescent molecule, a second oligonucleotide, and a second quencher molecule connected in sequence; and a third amplification primer pair is used for PCR amplification of a reference gene in the cfDNA sample, and a third fluorescent label comprising a third luminescent molecule, a third oligonucleotide, and a third quencher molecule connected in sequence; The PCR mentioned above is methylation-specific quantitative real-time PCR; The first amplification primer pair includes a first upstream primer and a first downstream primer, the nucleotide sequence of the first upstream primer is shown in SEQ ID NO.3, and the nucleotide sequence of the first downstream primer is shown in SEQ ID NO.4; The second amplification primer pair includes a second upstream primer and a second downstream primer, the nucleotide sequence of the second upstream primer is shown in SEQ ID NO.5, and the nucleotide sequence of the second downstream primer is shown in SEQ ID NO.6; The third amplification primer pair includes a third upstream primer and a third downstream primer. The nucleotide sequence of the third upstream primer is shown in SEQ ID NO.9, and the nucleotide sequence of the third downstream primer is shown in SEQ ID NO.

10. The nucleotide sequence of the first oligonucleotide is shown in SEQ ID NO.7, the nucleotide sequence of the second oligonucleotide is shown in SEQ ID NO.8, and the nucleotide sequence of the third oligonucleotide is shown in SEQ ID NO.

11.

2. The reagent kit according to claim 1, characterized in that, The first luminescent molecule and the second luminescent molecule are each independently selected from FAM, Cy5, VIC, TET, HEX, JOE, ROX; and / or The first luminescent molecule and the second luminescent molecule are different types of luminescent molecules; and / or The cfDNA samples were obtained from blood, plasma, or serum.

3. The reagent kit according to claim 1, characterized in that, The kit also includes: The third fluorescent label consists of a third luminescent molecule, a third oligonucleotide, and a third quencher molecule linked together in sequence.

4. The reagent kit according to claim 3, characterized in that, The third luminescent molecule is selected from FAM, Cy5, VIC, TET, HEX, JOE, ROX; and / or The third luminescent molecule is a different type of luminescent molecule from the first and second luminescent molecules.

5. The reagent kit according to claim 1, characterized in that, The kit further includes: extraction reagents for extracting the cfDNA sample from an in vitro sample; and / or The conversion reagent includes bisulfite; and / or The PCR amplification reagent is used to perform PCR amplification of the first transformed DNA strand A using the first amplification primer pair and the first fluorescent label, but not to perform PCR amplification of the first transformed DNA strand B; and / or, the second transformed DNA strand A is used to perform PCR amplification of the second transformed DNA strand A using the second amplification primer pair and the second fluorescent label, but not to perform PCR amplification of the second transformed DNA strand B.

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