Biomarkers, kits and methods of use for detecting early liver cancer

Abstract

The embodiments of the present application disclose a biomarker, a kit and a method of using the same for detecting early liver cancer. The kit of the embodiment of the present application uses a first DNA molecule as shown in SEQ ID NO.1 and / or a second DNA molecule as shown in SEQ ID NO.2 as a detection marker, and determines whether the in vitro sample is a positive sample for early liver cancer by judging the degree of methylation of the CpG site in the first DNA molecule and / or the second DNA molecule in the in vitro sample, thereby indicating whether the organism derived from the in vitro sample has early liver cancer lesions. The kit of the embodiment of the present application has good specificity and sensitivity for positive judgment of early liver cancer based on its specific biomarker, and can be used for screening of early liver cancer.

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and in particular to a biomarker, a kit and a method for using the same for detecting early liver cancer. Background Art

[0002] Liver cancer is one of the most common malignant tumors in China. Most liver cancer patients are already in the middle or advanced stages when they are discovered, with a median survival of only 1 to 1.5 years. However, the 5-year survival rate of early liver cancer after surgery is over 70%. Therefore, early screening, early diagnosis, and early treatment of liver cancer can greatly improve the prognosis of patients.

[0003] The blood marker currently used for liver cancer detection is alpha fetoprotein (AFP). However, AFP has problems with insufficient sensitivity and specificity in detecting early liver cancer. Summary of the invention

[0004] The embodiments of the present application provide a biomarker, a kit and a method of using the same for detecting early liver cancer, which can solve the problem of insufficient sensitivity and specificity when using alpha-fetoprotein to detect early liver cancer.

[0005] The present application embodiment provides a use of a separated biomarker or a combination of biomarkers as a biomarker for early liver cancer, wherein the biomarker comprises: a first DNA molecule as shown in SEQ ID NO.1 and / or a second DNA molecule as shown in SEQ ID NO.2. The first DNA molecule and the second DNA molecule include not only the base sequence information of DNA, but also naturally occurring DNA fragment substances. The first DNA molecule and the second DNA molecule each independently include methylated CpG sites and / or unmethylated CpG sites.

[0006] Alternatively, in some embodiments, all CpG sites of the first DNA molecule and / or the second DNA molecule used as a biomarker are methylated CpG sites. In other embodiments, all CpG sites of the first DNA molecule and / or the second DNA molecule used as a biomarker are non-methylated CpG sites.

[0007] The present application embodiment provides a use of a separated biomarker or a biomarker combination as a detection object of a kit for determining whether an ex vivo sample is an early liver cancer positive sample, and the biomarker combination includes: a first DNA molecule as shown in SEQ ID NO.1 and / or a second DNA molecule as shown in SEQ ID NO.2. The first DNA molecule and the second DNA molecule each independently include a methylated CpG site and an unmethylated CpG site.

[0008] Alternatively, in some embodiments, all CpG sites of the first DNA molecule and / or the second DNA molecule used as a biomarker are methylated CpG sites. In other embodiments, all CpG sites of the first DNA molecule and / or the second DNA molecule used as a biomarker are non-methylated CpG sites.

[0009] Alternatively, in some embodiments, the first DNA molecule is selected from the DOK1 gene with genomic coordinates of chr2:74555005-74555272. The second DNA molecule is selected from the PITX1 gene with genomic coordinates of chr5:135027454-135027645.

[0010] The present application embodiment provides a kit for identifying positive samples of early liver cancer. The kit indicates early liver cancer by identifying whether a certain ex vivo sample is a positive sample of early liver cancer. The kit includes: a conversion reagent, a first primer pair and a first fluorescent probe. In other embodiments, the kit may also include a second primer pair and a second fluorescent probe.

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

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

[0013] In some embodiments, the first primer pair is used for PCR amplification of the first converted DNA molecule A. The first fluorescent probe includes a first fluorescent group, a first DNA probe and a first quenching group connected in sequence. After the first DNA molecule containing a methylated CpG site is converted into the first converted DNA molecule A, the unmethylated cytosine is converted into uracil, but the methylated cytosine in the CpG site remains unchanged; after the first DNA molecule not containing a methylated CpG site is converted into the first converted DNA molecule B, all cytosines are converted into uracil. Therefore, the nucleotide sequence of the first converted DNA molecule A is different from that of the first converted DNA molecule B. The first primer pair and the first fluorescent probe in the embodiment of the present application are designed for the first converted DNA molecule A, so they can specifically amplify and bind to the first converted DNA molecule A, but will not amplify or bind to the first converted DNA molecule B.

[0014] In some embodiments, the second primer pair is used for PCR amplification of the second converted DNA molecule. The second fluorescent probe includes a second fluorescent group, a second DNA probe and a second quenching group connected in sequence. After the second DNA molecule containing a methylated CpG site is converted into a second converted DNA molecule A, unmethylated cytosine is converted into uracil, but the methylated cytosine in the CpG site remains unchanged; after the second DNA molecule not containing a methylated CpG site is converted into a second converted DNA molecule B, all cytosines are converted into uracil. Therefore, the nucleotide sequence of the second converted DNA molecule A is different from that of the second converted DNA molecule B. The second primer pair and the second fluorescent probe in the embodiment of the present application are designed for the second converted DNA molecule A, so it can specifically amplify and bind to the second converted DNA molecule A, but will not amplify or bind to the second converted DNA molecule B.

[0015] In some embodiments, in each cycle of PCR amplification of the cfDNA sample, the first DNA probe is correspondingly integrated into each newly amplified first converted DNA molecule A, and the first fluorescent group is separated from the first quenching group. After the first fluorescent group is separated from the first quenching group, the fluorescence emission of the first fluorescent group is not affected by the quenching effect of the first quenching group.

[0016] In some embodiments, in each cycle of PCR amplification of the cfDNA sample, the second DNA probe is correspondingly integrated into each newly amplified second converted DNA molecule A, and the second fluorescent group is separated from the second quenching group. After the second fluorescent group is separated from the second quenching group, the fluorescence emission of the second fluorescent group is not affected by the quenching effect of the second quenching group.

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

[0018] In some embodiments, the first fluorescent moiety and the second fluorescent moiety are different types of fluorescent moieties.

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

[0020] In some embodiments, the PCR is fluorescent quantitative PCR.

[0021] In some embodiments, the first DNA probe has a sequence identical or complementary to that of the first converted DNA molecule A having 10 to 25 consecutive nucleotides.

[0022] In some embodiments, the second DNA probe has a sequence identical or complementary to that of the second converted DNA molecule A having 6 to 15 consecutive nucleotides.

[0023] In some embodiments, the first primer pair includes a first forward primer as shown in SEQ ID NO.3 and a first reverse primer as shown in SEQ ID NO.4.

[0024] In some embodiments, the second primer pair includes a second forward primer as shown in SEQ ID NO.5 and a second reverse primer as shown in SEQ ID NO.6.

[0025] In some embodiments, the nucleotide sequence of the first DNA probe is shown as SEQ ID NO.7.

[0026] In some embodiments, the nucleotide sequence of the second DNA probe is shown as SEQ ID NO.8.

[0027] In some embodiments, the kit further comprises: a third primer pair and a third fluorescent probe. The third primer pair is used for PCR amplification of an internal reference gene in a cfDNA sample. The third fluorescent probe comprises a third fluorescent group, a third DNA probe and a third quenching group connected in sequence. The third DNA probe is identical or complementary to a portion of the sequence of the internal reference gene.

[0028] In some embodiments, the internal reference gene is selected from any one of the 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).

[0029] In some embodiments, the third primer pair includes a third forward primer as shown in SEQ ID NO.9 and a third reverse primer as shown in SEQ ID NO.10.

[0030] In some embodiments, the nucleotide sequence of the third DNA probe is shown as SEQ ID NO.11.

[0031] In some embodiments, in each cycle of PCR amplification of the cfDNA sample, the third DNA probe is integrated into each newly amplified internal reference gene one by one, and the third fluorescent group is separated from the third quenching group.

[0032] In some embodiments, the third fluorescent group is selected from FAM, Cy5, VIC, TET, HEX, JOE, ROX.

[0033] In some embodiments, the third fluorescent group is a different type of fluorescent group from the first fluorescent group and the second fluorescent group to avoid mutual interference of fluorescence when the three fluorescent groups coexist in the PCR reaction solution.

[0034] In some embodiments, the PCR is fluorescent quantitative PCR.

[0035] In some embodiments, the third DNA probe has 7 to 12 consecutive nucleotides that are identical or complementary to the internal reference gene.

[0036] In some embodiments, the kit further includes: extraction reagents and PCR amplification reagents, etc.

[0037] The extraction reagent is used to extract cfDNA samples from in vitro samples, which are plasma, serum or blood.

[0038] The PCR amplification reagent is used to perform PCR amplification on the first converted DNA molecule A using the first primer pair and the first fluorescent probe, and to perform PCR amplification on the second converted DNA molecule A using the second primer pair and the second fluorescent probe. When the third primer pair and the third fluorescent probe are also present in the PCR reaction solution, the PCR amplification reagent can also perform PCR amplification on the internal reference gene using the third primer pair and the third fluorescent probe.

[0039] In some embodiments, the method for determining whether a certain ex vivo sample is an early liver cancer positive sample in the kit of the present application is as follows:

[0040] The cfDNA sample is transformed using a transformation reagent, and then the above reagents are mixed to form a PCR reaction solution, and then PCR amplification is performed, and the fluorescence intensity of the PCR reaction solution is detected in real time. The PCR reaction solution includes at least the transformed DNA sample, the first primer pair, the first fluorescent probe, the second primer pair, the second fluorescent probe, and the PCR amplification reagent.

[0041] Under the condition that the fluorescence intensity of the first fluorescent group in the PCR reaction solution reaches or exceeds the fluorescence threshold and the amplification cycle number (Ct value) of the first converted DNA molecule A is greater than the first cycle threshold (such as 38 to 44), the first DNA molecule is judged as methylation negative, otherwise it is judged as methylation positive.

[0042] Under the condition that the fluorescence intensity of the second fluorescent group in the PCR reaction solution reaches or exceeds the fluorescence threshold and the amplification cycle number (Ct value) of the second transformed DNA molecule A is greater than the second cycle threshold (such as 38 to 44), the second transformed DNA molecule is judged as methylation negative, otherwise it is judged as methylation positive.

[0043] When either or both of the first DNA molecule and the second DNA molecule are determined to be methylation positive, the cfDNA sample is determined to be an early liver cancer positive sample, otherwise the cfDNA sample is determined to be an early liver cancer negative sample. Specifically, the following situations are included:

[0044] 1. When the first DNA molecule in the cfDNA sample is judged to be methylation-negative, and the second DNA molecule in the cfDNA sample is judged to be methylation-negative, the cfDNA sample is judged to be an early liver cancer-negative sample.

[0045] 2. When the first DNA molecule in the cfDNA sample is judged to be methylation-positive and the second converted DNA molecule in the cfDNA sample is judged to be methylation-negative, the cfDNA sample is judged to be an early liver cancer-positive sample.

[0046] 3. When the first DNA molecule in the cfDNA sample is judged to be methylation-negative, and the second converted DNA molecule in the cfDNA sample is judged to be methylation-positive, the cfDNA sample is judged to be an early liver cancer positive sample.

[0047] 4. When the first DNA molecule in the cfDNA sample is judged to be methylation-positive, and the second converted DNA molecule in the cfDNA sample is judged to be methylation-positive, the cfDNA sample is judged to be an early liver cancer-positive sample.

[0048] In other embodiments, the PCR reaction solution further includes a third primer pair and a third fluorescent probe. At this time, under the condition that the fluorescence intensity of the third fluorescent group in the PCR reaction solution reaches or exceeds the fluorescence threshold and the amplification cycle number (Ct value) of the internal reference gene is less than or equal to the third cycle threshold (such as 30 to 32), the current PCR amplification is determined to be effective amplification, and the current PCR amplification result can be trusted; otherwise, it is determined to be invalid amplification, and the current PCR amplification result cannot be trusted, and the PCR amplification needs to be repeated.

[0049] Some embodiments of the present application provide a method for using the above-mentioned kit, which comprises the following steps:

[0050] 1. Using a conversion reagent, converting unmethylated cytosine in a first DNA molecule containing a methylated CpG site in a cfDNA sample into uracil to obtain a first converted DNA molecule A, and converting all cytosine in a first DNA molecule not containing a methylated CpG site in a cfDNA sample into uracil to obtain a first converted DNA molecule B, thereby obtaining a first DNA sample; the nucleotide sequence of the first DNA molecule is shown in SEQ ID NO.1;

[0051] 2. Providing a first primer pair and a first fluorescent probe; the first primer pair is used for PCR amplification of the first converted DNA molecule A; the first fluorescent probe comprises a first fluorescent group, a first DNA probe and a first quenching group connected in sequence;

[0052] 3. Adding a first primer pair, a first fluorescent probe and a PCR amplification reagent to the first DNA sample to obtain a first PCR reaction solution, and performing PCR amplification;

[0053] 4. Detecting the fluorescence intensity of the first PCR reaction solution, and recording the required number of PCR amplification cycles after the fluorescence intensity of the first fluorescent group in the first PCR reaction solution reaches or exceeds the fluorescence threshold;

[0054] 5. If the number of amplification cycles of the first converted DNA molecule A is greater than the first cycle threshold, the first DNA molecule is determined to be methylation negative, otherwise it is determined to be methylation positive;

[0055] When the first DNA molecule is determined to be methylation positive, the cfDNA sample is determined to be an early liver cancer positive sample, otherwise the cfDNA sample is determined to be an early liver cancer negative sample.

[0056] Some embodiments of the present application provide a method for using the above-mentioned kit, which includes using an extraction reagent to process an in vitro sample to obtain a cfDNA sample.

[0057] Some embodiments of the present application provide methods for using the above-mentioned kit, further comprising:

[0058] The unmethylated cytosine in the first DNA molecule containing the methylated CpG site in the cfDNA sample is converted into uracil using a conversion reagent to obtain a first converted DNA molecule A, and all the cytosines in the first DNA molecule without the methylated CpG site in the cfDNA sample are converted into uracil to obtain a first converted DNA molecule B, thereby obtaining a first DNA sample; the nucleotide sequence of the first DNA molecule is shown in SEQ ID NO. 1. The unmethylated cytosine in the second DNA molecule containing the methylated CpG site in the cfDNA sample is converted into uracil to obtain a second converted DNA molecule A, and all the cytosines in the second DNA molecule without the methylated CpG site in the cfDNA sample are converted into uracil to obtain a second converted DNA molecule B, thereby obtaining a second DNA sample; the nucleotide sequence of the second DNA molecule is shown in SEQ ID NO. 2.

[0059] 3. Provide a first primer pair, a first fluorescent probe, a second primer pair, and a second fluorescent probe. The first primer pair is used for PCR amplification of the first converted DNA molecule A. The first fluorescent probe includes a first fluorescent group, a first DNA probe, and a first quenching group connected in sequence. The second primer pair is used for PCR amplification of the second converted DNA molecule A. The second fluorescent probe includes a second fluorescent group, a second DNA probe, and a second quenching group connected in sequence.

[0060] 4. Add the first primer pair, the first fluorescent probe, the second primer pair, the second fluorescent probe and the PCR amplification reagent to the converted DNA sample (containing the first DNA sample and the second DNA sample mentioned above) to obtain a second PCR reaction solution, and perform PCR amplification.

[0061] 5. Detect the fluorescence intensity of the second PCR reaction solution. After the fluorescence intensity of the first fluorescent group and the fluorescence intensity of the second fluorescent group in the second PCR reaction solution reach or exceed the fluorescence threshold, record the required number of PCR amplification cycles.

[0062] 6. If the number of amplification cycles of the first converted DNA molecule A is greater than the first cycle threshold, the first DNA molecule is judged as methylation negative, otherwise it is judged as methylation positive; if the number of amplification cycles of the second converted DNA molecule A is greater than the second cycle threshold, the second DNA molecule is judged as methylation negative, otherwise it is judged as methylation positive;

[0063] When either or both of the first DNA molecule and the second DNA molecule are determined to be methylation positive, the cfDNA sample is determined to be an early liver cancer positive sample, otherwise the cfDNA sample is determined to be an early liver cancer negative sample. The specific determination method is detailed in the description of the kit and will not be repeated here.

[0064] In some embodiments, the first cycle threshold is 38-44.

[0065] In some embodiments, the second cycle threshold is 38-44.

[0066] In some embodiments, the method of using the kit further comprises the following steps:

[0067] 1. Providing a third primer pair and a third fluorescent probe; the third primer pair is used for PCR amplification of an internal reference gene in a cfDNA sample; the third fluorescent probe comprises a third fluorescent group, a third DNA probe and a third quenching group connected in sequence;

[0068] 2. Adding the third primer pair and the third fluorescent probe to the first PCR reaction solution or the second PCR reaction solution to perform PCR amplification;

[0069] 3. After the fluorescence intensity of the third fluorescent group reaches or exceeds the fluorescence threshold, record the required number of PCR amplification cycles;

[0070] 4. When the number of amplification cycles of the internal reference gene is less than or equal to the third cycle threshold, the PCR amplification is determined to be effective amplification, otherwise it is determined to be invalid amplification.

[0071] In some embodiments of the present application, the kit includes a reagent that can specifically detect the methylation level of the DNA molecules shown in SEQ ID NO.1 and SEQ ID NO.2.

[0072] In some embodiments of the present application, the kit further comprises a positive reference. All CpG sites in the nucleotide sequence of the above-mentioned biomarker are methylated to become the above-mentioned positive reference, and therefore, all CpG sites in the positive reference are methylated CpG sites.

[0073] In some embodiments of the present application, the kit further comprises a negative reference. All CpG sites in the nucleotide sequence of the above-mentioned biomarker are demethylated or unmethylated to become the above-mentioned negative reference, therefore, all CpG sites in the negative reference are unmethylated CpG sites, which do not include any methylated CpG sites.

[0074] The biomarkers provided in the embodiments of the present application can be used as positive references and negative references for detecting early liver cancer, and can effectively indicate the test results and correct the accuracy of the test results.

[0075] By adopting the technical solution in any of the above embodiments, the present application achieves the following beneficial effects:

[0076] The kit of the embodiment of the present application uses the first DNA molecule shown in SEQ ID NO.1 and the second DNA molecule shown in SEQ ID NO.2 as detection markers. The embodiment of the present application uses a conversion reagent to convert the first DNA molecule containing a methylated CpG site in the cfDNA sample into a first converted DNA molecule A, and / or, converts the second DNA molecule containing a methylated CpG site in the cfDNA sample into a second converted DNA molecule A. Fluorescence quantitative PCR is used to determine the degree of methylation of the CpG site in the first DNA molecule and / or the second DNA molecule, and then determine whether the cfDNA sample is a positive sample for early liver cancer, thereby indicating whether the organism derived from the in vitro sample has early liver cancer lesions. The kit of the embodiment of the present application has good specificity and sensitivity for positive judgment of early liver cancer based on its specific biomarkers, and can be used for screening of early liver cancer. DETAILED DESCRIPTION

[0077] The technical solutions in the embodiments of the present application are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present application.

[0078] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments. In addition, in the description of this application, the term "including" means "including but not limited to". Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and simplicity and should not be understood 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 the range.

[0079] The present application embodiment provides a use of a separated biomarker or a combination of biomarkers as a biomarker for early liver cancer. The biomarker includes: a first DNA molecule as shown in SEQ ID NO.1 and / or a second DNA molecule as shown in SEQ ID NO.2. The first DNA molecule and the second DNA molecule each independently include a methylated CpG site and an unmethylated CpG site.

[0080] Optionally, in some embodiments, all CpG sites of the first DNA molecule and / or the second DNA molecule used for the biomarker are methylated CpG sites. In other embodiments, all CpG sites of the first DNA molecule and / or the second DNA molecule used for the biomarker are non-methylated CpG sites.

[0081] Experiments have shown that the methylation level of the CpG sites of the first and second DNA molecules in the subject's cfDNA sample can be used to determine whether the subject is an early liver cancer positive patient or an early liver cancer negative patient. If the subject is determined to be an early liver cancer positive patient, further judgment can be made in combination with histological testing, imaging testing or other physical and chemical indicators.

[0082] The first DNA molecule corresponds to the DOK1 gene, and its genomic coordinates are chr2:74555005-74555272. The second DNA molecule corresponds to the PITX1 gene, and its genomic coordinates are chr5:135027454-135027645. The genomic coordinates are all the hg38 version of the human reference genome coordinates.

[0083] In some embodiments of the present application, an "isolated" biomarker refers to a biomarker that is extracted, separated, and purified from an individual or biological sample, or a biomarker that is synthesized by chemical / biological methods known in the art, and only needs to have a nucleotide sequence.

[0084] In an embodiment of the present application, the biomarker can be synthesized to become a nucleotide sequence in which all CpG sites are methylated, so that the biomarker can be used as a positive reference. In other embodiments, the biomarker combination can be synthesized to become a nucleotide sequence including all methylated CpG sites, so that the biomarker combination can be used as a positive reference.

[0085] In the embodiments of the present application, "methylation" is a form of DNA chemical modification, which can change genetic expression without changing the DNA sequence. DNA methylation refers to the covalent binding of a methyl group to the 5th carbon position of the cytosine of the genomic CpG dinucleotide under the action of DNA methyltransferase. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability and the way DNA interacts with proteins, thereby controlling gene expression. Methylation of mammalian DNA generally occurs on cytosine in cytosine-phosphate-guanine (CpG) dinucleotide sites, showing 5-methylcytosine (5mC), while cytosine in non-CpG sites is generally not modified by methylation. After DNA is treated with bisulfite, non-methylated cytosine in DNA will be converted into uracil, while methylated cytosine remains unchanged. Therefore, in general, 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 non-methylated cytosines are converted to uracil; if a DNA molecule does not contain methylated CpG, then all cytosines are converted to uracil because they are not methylated. This uracil pairs with adenine during the PCR process.

[0086] In the embodiments of the present 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 in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. Typical deoxyribonucleotides of DNA are thymine, adenine, cytosine, and guanine. Typical ribonucleotides of RNA are uracil, adenine, cytosine, and guanine.

[0087] The liver cancer in the embodiments of the present application specifically refers to hepatocellular carcinoma.

[0088] The embodiments of the present application provide a use of an isolated biomarker or a combination of biomarkers in preparing a kit for detecting early liver cancer, wherein the biomarker includes: a first DNA molecule as shown in SEQ ID NO.1 and / or a second DNA molecule as shown in SEQ ID NO.2.

[0089] In some embodiments, all CpG sites of the first DNA molecule and / or the second DNA molecule used as biomarkers are methylated CpG sites. A nucleotide sequence in which all CpG sites are methylated can be used as a positive reference.

[0090] In some embodiments, all CpG sites of the first DNA molecule and / or the second DNA molecule used as biomarkers are unmethylated CpG sites, and nucleotide sequences in which all CpG sites are unmethylated can be used as negative references.

[0091] The present application embodiment provides a use of a separated biomarker combination as a detection object of a kit for determining whether an in vitro sample is an early liver cancer positive sample, and the biomarker combination includes: a first DNA molecule as shown in SEQ ID NO.1 and a second DNA molecule as shown in SEQ ID NO.2. The first DNA molecule and the second DNA molecule each independently include a methylated CpG site and an unmethylated CpG site, or all CpG sites of the first DNA molecule and / or the second DNA molecule are methylated CpG sites, or all CpG sites of the first DNA molecule and / or the second DNA molecule are unmethylated CpG sites.

[0092] The present application provides a method for using the above kit, which comprises the following steps:

[0093] 1. Use a conversion reagent to convert unmethylated cytosine in the first DNA molecule containing a methylated CpG site in the cfDNA sample into uracil to obtain a first converted DNA molecule A; convert all cytosine in the first DNA molecule without a methylated CpG site in the cfDNA sample into uracil to obtain a first converted DNA molecule B, thereby obtaining a first DNA sample. The nucleotide sequence of the first DNA molecule is shown in SEQ ID NO.1. The conversion reagent can be a Zymo bisulfite conversion kit.

[0094] 2. Provide a first primer pair and a first fluorescent probe; the first primer pair is used for PCR amplification of the first converted DNA molecule A. The first fluorescent probe comprises a first fluorescent group, a first DNA probe and a first quenching group connected in sequence.

[0095] 3. Add a first primer pair, a first fluorescent probe and a PCR amplification reagent to the first DNA sample to obtain a first PCR reaction solution, and perform PCR amplification.

[0096] 4. Detecting the fluorescence intensity of the first PCR reaction solution, and recording the required number of PCR amplification cycles after the fluorescence intensity of the first fluorescent group in the first PCR reaction solution reaches or exceeds the fluorescence threshold;

[0097] Under the condition that the amplification cycle number of the first converted DNA molecule A is greater than the first cycle threshold, the first DNA molecule is determined to be methylation negative, otherwise it is determined to be methylation positive;

[0098] When the first DNA molecule is determined to be methylation positive, the cfDNA sample is determined to be an early liver cancer positive sample, otherwise the cfDNA sample is determined to be an early liver cancer negative sample.

[0099] The present application embodiment provides a kit for detecting early liver cancer, which is used to identify whether an in vitro sample is an early liver cancer positive sample. The kit includes: a first primer pair, a first fluorescent probe, a second primer pair and a second fluorescent probe.

[0100] Wherein, the first primer pair is used for PCR amplification of the first transformed DNA molecule A. The PCR in the embodiment of the present application can be methylation-specific fluorescent quantitative PCR. In the exponential period of PCR amplification, the Ct value (Cycle threshold, cycle threshold) of the amplified DNA, the final fluorescence intensity and the logarithm of the starting copy number of the DNA have a linear relationship, so relative quantification can be performed.

[0101] The first fluorescent probe includes a first fluorescent group, a first DNA probe and a first quenching group connected in sequence. The first fluorescent group can be selected from fluorescent groups such as FAM, Cy5, VIC, TET, HEX, JOE, ROX, etc. The properties of the above-mentioned fluorescent groups are shown in Table 1 below.

[0102] Table 1 Properties of various fluorescent groups

[0103]

[0104]

[0105] The first quenching group can be selected from BHQ-1 carboxylic acid, BHQ-2 carboxylic acid, BHQ-3 carboxylic acid, carboxytetramethylrhodamine (TAMRA) and 4-(4-oxaalkylaminophenylazo)benzoic acid (DABCYL). For example, the quenching group of VIC can be BHQ1, and the quenching group of Cy5 can be BHQ2.

[0106] In the first fluorescent probe, the first fluorescent group is connected to the 5' end of the first DNA probe, and the first quencher group is connected to the 3' end of the first DNA probe. When the first fluorescent probe is intact, the fluorescent signal emitted by the first fluorescent group is absorbed by the first quencher group, so that the first fluorescent probe does not emit a fluorescent signal. During the exponential phase of methylation-specific fluorescent quantitative PCR amplification, the 5'-3' exonuclease activity of the Taq enzyme enzymatically degrades the first fluorescent probe, so that the first fluorescent group is separated from the first quencher group, and the sequence in the first DNA probe that is identical or complementary to the first converted DNA molecule A is correspondingly integrated into each newly amplified first converted DNA molecule A. Therefore, when each first fluorescent probe is enzymatically degraded and a part of the related sequence is integrated into each newly amplified first converted DNA molecule A, a first fluorescent group will be released. At this time, the first fluorescent group is separated from the first quencher group, and its fluorescence will not be quenched by the first quencher group. Therefore, the number and fluorescence intensity of the first fluorescent group are linearly related to the amplified first converted DNA molecule A, thereby achieving quantification. The first DNA probe is identical or complementary to a portion of the sequence of the first transformed DNA molecule A. Therefore, in the exponential period of PCR amplification, that is, in each cycle of PCR amplification of the DNA sample, the sequence identical or complementary to the first transformed DNA molecule A in the first DNA probe is integrated into each newly amplified first transformed DNA molecule A. The first primer pair and the first fluorescent probe in the embodiment of the present application are designed for the first transformed DNA molecule A, so they can specifically amplify and bind to the first transformed DNA molecule A, but will not amplify or bind to the first transformed DNA molecule B.

[0107] The second primer pair is used to PCR amplify the second converted DNA molecule A in the converted cfDNA sample.

[0108] The second fluorescent probe includes a second fluorescent group, a second DNA probe and a second quenching group connected in sequence. The second fluorescent group can be selected from fluorescent groups such as FAM, Cy5, VIC, TET, HEX, JOE, ROX, etc., but the second fluorescent group and the first fluorescent group are different types of fluorescent groups to avoid mutual interference between the fluorescence emitted by different fluorescent groups. The second quenching group can be selected from BHQ-1 carboxylic acid, BHQ-2 carboxylic acid, BHQ-3 carboxylic acid, carboxytetramethylrhodamine (TAMRA) and 4-(4-oxanylaminophenylazo)benzoic acid (DABCYL). In the second fluorescent probe, the second fluorescent group is connected to the 5' end of the second DNA probe, and the second quenching group is connected to the 3' end of the second DNA probe. The mechanism of action of the second fluorescent probe can refer to the first fluorescent probe. The second DNA probe is identical or complementary to a portion of the sequence of the second converted DNA molecule A, so that the sequence identical or complementary to the second converted DNA molecule A in the second DNA probe in each cycle of PCR amplification of the converted cfDNA sample is integrated into each newly amplified second converted DNA molecule A one-to-one. The second primer pair and the second fluorescent probe in the embodiment of the present application are designed for the second transformed DNA molecule A, so they can specifically amplify and bind to the second transformed DNA molecule A, but will not amplify or bind to the second transformed DNA molecule B.

[0109] In an embodiment of the present application, the DNA sample is from blood, serum or plasma. For example, the DNA sample can be cell-free DNA (cfDNA). cfDNA is fragmented DNA in human peripheral blood and other circulating body fluids.

[0110] cfDNA can exist mainly in the form of entangled nucleosomes in the blood, because in this case, cfDNA not entangled in nucleosomes will be quickly degraded. The cfDNA entangled in a nucleosome is about 170bp, so the main fragment of cfDNA has a main peak at 170bp (equivalent to one nucleosome), and then a small peak at 340bp (equivalent to two nucleosomes), and so on.

[0111] In some other embodiments, the length of cfDNA may also be between 170 bp and 210 bp (including this number). However, in some other embodiments, the length of cfDNA may also be 180 bp, 185 bp, 190 bp, 195 bp, 200 bp, etc.

[0112] In some other embodiments, the length of cfDNA can be an integer multiple of any value between 170 bp and 210 bp (determined according to the number of entangled nucleosomes), such as 340 bp, 420 bp, etc.

[0113] Studies have shown that in the plasma of cancer patients, cfDNA can carry mutations and epigenetic aberrations of tumor DNA, especially abnormal DNA methylation changes in the early stages of liver cancer. DNA methylation in mammals mainly occurs on cytosine in cytosine-phosphate-guanine (CpG) dinucleotides, expressed as 5-methylcytosine (5mC). Compared with normal liver tissue, global hypomethylation of genomic DNA and local or global hypermethylation of DNA located in CpG islands often occur in liver cancer tissues. Abnormal hypermethylation of CpG islands can regulate the expression of cancer-related genes, thereby playing an important role in the occurrence and development of liver cancer. The inventors found that abnormal methylation levels of DOK1 and PITX1 genes are closely related to early liver cancer, which is manifested in the plasma of patients with early liver cancer. The related sequences of DOK1 and PITX1 genes are abnormally methylated, while in the plasma of non-liver cancer patients, the related sequences of DOK1 and PITX1 genes are not methylated. Therefore, the embodiment of the present application detects early liver cancer by detecting the methylation levels of specific sequences of the DOK1 gene and the PITX1 gene in the cfDNA sample.

[0114] In the embodiment of the present application, the first primer pair includes a first forward primer as shown in SEQ ID NO.3 and a first reverse primer as shown in SEQ ID NO.4. The second primer pair includes a second forward primer as shown in SEQ ID NO.5 and a second reverse primer as shown in SEQ ID NO.6.

[0115] In the embodiment of the present application, the nucleotide sequence of the first DNA probe is shown as SEQ ID NO. 7. This nucleotide sequence can be embedded in each newly amplified first converted DNA molecule A in each cycle of PCR amplification, and the first fluorescent group and the first quenching group will also be separated as the structural integrity of the first fluorescent probe is destroyed.

[0116] In the embodiment of the present application, the nucleotide sequence of the second DNA probe is shown in SEQ ID NO. 8. This nucleotide sequence can be embedded in each newly amplified second converted DNA molecule A in each cycle of PCR amplification, and the second fluorescent group and the second quenching group will also separate as the structural integrity of the second fluorescent probe is destroyed.

[0117] In an embodiment of the present application, the kit further includes: a third primer pair and a third fluorescent probe.

[0118] Among them, the third primer pair is used for PCR amplification of the internal reference gene in the cfDNA sample. The internal reference gene is selected from any one of the 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 primer pair includes a third forward primer as shown in SEQ ID NO.9 and a third reverse primer as shown in SEQ ID NO.10. Among them, the genomic coordinates of the ACTB gene-related region are chr7:5529432-5529682, and its nucleotide sequence is shown in SEQ ID NO.12.

[0119] The third fluorescent probe includes a third fluorescent group, a third DNA probe and a third quenching group connected in sequence. The third fluorescent group can be selected from fluorescent groups such as FAM, Cy5, VIC, TET, HEX, JOE, ROX, etc., but the third fluorescent group is a fluorescent group of a different type from the first fluorescent group and the second fluorescent group, so as to interfere with each other in fluorescence. The third quenching group can be selected from BHQ-1 carboxylic acid, BHQ-2 carboxylic acid, BHQ-3 carboxylic acid, carboxytetramethylrhodamine (TAMRA) and 4-(4-oxaalkylaminophenylazo)benzoic acid (DABCYL). The mechanism of action of the third fluorescent group and the third quenching group refers to the first fluorescent group and the first quenching group. The nucleotide sequence of the third DNA probe is shown in SEQ ID NO.11. This segment of base sequence can be embedded in each newly amplified internal reference gene in each cycle of PCR amplification, and the third fluorescent group and the third quenching group will also be separated as the structural integrity of the third fluorescent probe is destroyed.

[0120] In some embodiments, the kit further includes: extraction reagents and / or PCR amplification reagents, etc.

[0121] The extraction reagent is used to extract cfDNA samples from in vitro samples. The in vitro samples can be plasma, serum or blood. The QIAamp Circulating Nucleic Acid Kit (Qiagen) is used as an extraction reagent to extract cfDNA from serum samples.

[0122] The PCR amplification system includes 25 μL of Taq Pro HS Probe Master Mix. The amount of converted cfDNA sample added is 10 μL. The reaction conditions of PCR are: first run at 95°C for 120 seconds for pre-denaturation; then run at 95°C for 10 seconds, 60°C for 30 seconds, for a total of 50 cycles, and finally store at 4°C. When the third primer pair and the third fluorescent probe are still present in the PCR reaction solution, the PCR amplification reagent can also use the third primer pair and the third fluorescent probe to amplify the internal reference gene in the cfDNA sample.

[0123] In some embodiments, the present application also provides a method for using the kit, the method comprising the following steps:

[0124] 1.1 Using a conversion reagent, convert unmethylated cytosine in a first DNA molecule containing a methylated CpG site in a cfDNA sample into uracil to obtain a first converted DNA molecule A, and convert all cytosine in the first DNA molecule without a methylated CpG site in the cfDNA sample into uracil to obtain a first converted DNA molecule B, thereby obtaining a first DNA sample. Using a conversion reagent, convert unmethylated cytosine in a second DNA molecule containing a methylated CpG site in the cfDNA sample into uracil to obtain a second converted DNA molecule A, and convert all cytosine in the second DNA molecule without a methylated CpG site in the cfDNA sample into uracil to obtain a second converted DNA molecule B, thereby obtaining a second DNA sample;

[0125] 1.2 providing a first primer pair, a first fluorescent probe, a second primer pair and a second fluorescent probe; the first primer pair is used for PCR amplification of a first converted DNA molecule A; the first fluorescent probe comprises a first fluorescent group, a first DNA probe and a first quenching group connected in sequence; the second primer pair is used for PCR amplification of a second converted DNA molecule A; the second fluorescent probe comprises a second fluorescent group, a second DNA probe and a second quenching group connected in sequence;

[0126] 1.3 Add the first primer pair, the first fluorescent probe, the second primer pair, the second fluorescent probe and the PCR amplification reagent to the converted DNA sample (including the first DNA sample and the second DNA sample) to obtain a second PCR reaction solution, and perform PCR amplification. Detect the fluorescence intensity of the second PCR reaction solution, and after the fluorescence intensity of the first fluorescent group and the fluorescence intensity of the second fluorescent group in the second PCR reaction solution reach or exceed the fluorescence threshold, record the required number of PCR amplification cycles;

[0127] 1.4 If the number of amplification cycles of the first converted DNA molecule A is greater than the first cycle threshold, the first DNA molecule is judged as methylation negative, otherwise it is judged as methylation positive; if the number of amplification cycles of the second converted DNA molecule A is greater than the second cycle threshold, the second DNA molecule is judged as methylation negative, otherwise it is judged as methylation positive. If either the first DNA molecule or the second DNA molecule is judged as methylation positive, the cfDNA sample is judged as an early liver cancer positive sample, otherwise the cfDNA sample is judged as an early liver cancer negative sample.

[0128] The first cycle threshold value may be 38 to 44, for example, 38, 39, 40, 41, 42, 43, or 44.

[0129] The second cycle threshold value may be 38 to 44, for example, 38, 39, 40, 41, 42, 43, or 44.

[0130] In some embodiments, the method of using the kit includes the following steps: using an extraction reagent to treat an in vitro sample to obtain the above-mentioned cfDNA sample.

[0131] In some embodiments, the method of using the kit comprises the following steps:

[0132] 3.1 providing a third primer pair and a third fluorescent probe; the third primer pair is used for PCR amplification of an internal reference gene in a DNA sample; the third fluorescent probe comprises a third fluorescent group, a third DNA probe and a third quenching group connected in sequence;

[0133] 3.2 Add the third primer pair and the third fluorescent probe together with the PCR amplification reagent to the converted cfDNA sample for PCR amplification; after the fluorescence intensity of the third fluorescent group reaches or exceeds the fluorescence threshold, record the required number of PCR amplification cycles;

[0134] 3.3 When the number of amplification cycles of the internal reference gene is less than or equal to the third cycle threshold, the PCR amplification is judged as effective amplification, otherwise it is judged as invalid amplification.

[0135] Among them, step 3.3 is used to determine whether a certain PCR amplification product can be trusted. If it can be trusted, it means that the PCR amplification result is valid and subsequent judgment work can be carried out. If it cannot be trusted, it means that the PCR amplification result is invalid and a new PCR amplification is required.

[0136] In the method of using the kit in the above embodiment, 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 of the above genes refers to the DOK1 gene corresponding to the first DNA molecule, the PITX1 gene corresponding to the second DNA molecule, and the ACTB gene corresponding to the internal reference gene. However, the internal reference gene is not limited to the ACTB gene.

[0137] In some embodiments, the fluorescence thresholds corresponding to the three fluorescent groups may be the same or different. For example, the fluorescence value of the first fluorescent group may correspond to the first fluorescence threshold, the fluorescence value of the second fluorescent group may correspond to the second fluorescence threshold, and the fluorescence value of the third fluorescent group may correspond to the third fluorescence threshold. In this case, the specific data of the first fluorescence threshold, the second fluorescence threshold, and the third fluorescence threshold are different.

[0138] The present application is further described below in conjunction with specific embodiments.

[0139] The in vitro samples used in the various embodiments of the present application include: blood samples from 100 healthy people (without liver cancer), blood samples from 150 patients with hepatitis B and liver cirrhosis (without liver cancer), and blood samples from 150 patients with liver cancer. The blood samples are stored in a free DNA special blood collection tube (Streck), which contains anticoagulants and cell stabilizers. Then, the kits in the embodiments of the present application are used to process the above in vitro samples according to the following methods.

[0140] The method of this embodiment includes the following steps:

[0141] 1. Use extraction reagent to process the ex vivo sample (i.e., blood sample) to obtain a cfDNA sample. This step specifically includes:

[0142] The above-mentioned in vitro samples (blood samples) were centrifuged to obtain plasma samples. The cfDNA samples in the plasma samples were extracted using the QIAamp Circulating Nucleic Acid Kit (Qiagen). The extraction method was referred to the instruction manual of the kit.

[0143] 2. Treat the cfDNA sample with a conversion reagent to convert unmethylated cytosine in the cfDNA sample into uracil to obtain a converted cfDNA sample (containing a first converted DNA molecule A, a first converted DNA molecule B, a second converted DNA molecule A, and a second converted DNA molecule B). This step specifically includes:

[0144] Take 10 ng of cfDNA from the cfDNA sample obtained in step 1 and convert the cfDNA using the Zymo bisulfite conversion kit. The conversion process converts all non-methylated cytosine in the cfDNA sample into uracil, while methylated cytosine remains unchanged.

[0145] 3. Provide a first primer pair, a first fluorescent probe, a second primer pair, a second fluorescent probe, a third primer pair, a third fluorescent probe and a PCR amplification reagent, as shown below:

[0146] The first primer pair includes a first forward primer as shown in SEQ ID NO. 3 and a first reverse primer as shown in SEQ ID NO. 4. The first primer pair is used for PCR amplification of a first converted DNA molecule A in a converted cfDNA sample. The first DNA molecule is derived from the DOK1 gene.

[0147] The first fluorescent probe comprises a first fluorescent group, a first DNA probe and a first quenching group connected in sequence. The first fluorescent group is FAM. The nucleotide sequence of the first DNA probe is shown in SEQ ID NO.7, which is reverse complementary to the first converted DNA molecule A. The first quenching group is BHQ-3.

[0148] The second primer pair includes a second forward primer as shown in SEQ ID NO. 5 and a second reverse primer as shown in SEQ ID NO. 6. The second primer pair is used for PCR amplification of the second transformed DNA molecule A. The second DNA molecule is derived from the PITX1 gene.

[0149] The second fluorescent probe includes a second fluorescent group, a second DNA probe and a second quenching group connected in sequence. The second fluorescent group is Cy5. The nucleotide sequence of the second DNA probe is shown in SEQ ID NO.8, which is reverse complementary to the second converted DNA molecule A. The second quenching group is BHQ-2.

[0150] The third primer pair includes a third forward primer as shown in SEQ ID NO.9 and a third reverse primer as shown in SEQ ID NO.10. The third primer pair is used for PCR amplification of an internal reference gene, the internal reference gene is the ACTB gene, and its nucleotide sequence is shown in SEQ ID NO.12.

[0151] The third fluorescent probe includes a third fluorescent group, a third DNA probe and a third quenching group connected in sequence. The third fluorescent group is VIC. The nucleotide sequence of the third DNA probe is shown in SEQ ID NO.11, which is reverse complementary to the sequence of the internal reference gene after all Cs are converted to Ts. The third quenching group is BHQ-1.

[0152] PCR amplification reagents include Taq Pro HS Probe Master Mix, which contains DNA polymerase, dNTPs, and buffer.

[0153] 4. Add the first primer pair, the first fluorescent probe, the second primer pair, the second fluorescent probe, the third primer pair, the third fluorescent probe and the PCR amplification reagent to the DNA sample to obtain a PCR reaction solution and perform PCR amplification. Detect the fluorescence intensity of the obtained PCR reaction solution, and after the fluorescence value of the first fluorescent group, the fluorescence value of the second fluorescent group and the fluorescence value of the third fluorescent group all exceed their respective fluorescence thresholds, record the required number of PCR amplification cycles. Use a Bio-Rad fluorescent quantitative PCR instrument for PCR amplification and fluorescence detection. Among them, the fluorescence value of the first fluorescent group needs to exceed the first fluorescence threshold, the fluorescence value of the second fluorescent group needs to exceed the second fluorescence threshold, and the fluorescence value of the third fluorescent group needs to exceed the third fluorescence threshold.

[0154] The components of the PCR reaction solution are shown in Table 2 below.

[0155] Table 2 Components of PCR reaction solution

[0156]

[0157] In Table 2, the forward primer includes a first forward primer, a second forward primer, and a third forward primer. The reverse primer includes a first reverse primer, a second reverse primer, and a third reverse primer. The fluorescent probe includes a first fluorescent probe, a second fluorescent probe, and a third fluorescent probe. In this embodiment, the above-mentioned primers and fluorescent probes are mixed with the DNA sample for detection, because the first fluorescent group FAM is green light, the second fluorescent group Cy5 is red light, and the third fluorescent group VIC is yellow light. The luminescent colors of the three fluorescent groups are different and will not interfere with each other. Therefore, they can be mixed together for detection. In other embodiments, the first primer pair and the first fluorescent probe, the second primer pair and the second fluorescent probe, the third primer pair and the third fluorescent probe can also be mixed with the converted cfDNA sample and the PCR amplification reagent, respectively, and detected separately.

[0158] The conditions for the PCR reaction are shown in Table 3 below.

[0159] Table 3 PCR reaction conditions

[0160]

[0161] 5. When the number of amplification cycles (Ct value) of the first converted DNA molecule A (corresponding to the DOK1 gene) is greater than the first cycle threshold (value of 40), the first DNA molecule is judged as methylation negative, otherwise it is judged as methylation positive. Because methylated cfDNA comes 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. The lower the degree of methylation in the cfDNA sample, the larger the Ct value. Therefore, the degree of methylation can be judged based on the Ct value.

[0162] Under the condition that the amplification cycle number (Ct value) of the second converted DNA molecule A (corresponding to the PITX1 gene) is greater than the second cycle threshold value (value of 40), the second DNA molecule is judged to be methylation negative, otherwise it is judged to be methylation positive. When any one of the first DNA molecule and the second DNA molecule is judged to be methylation positive, the DNA sample is judged to be an early liver cancer positive sample, otherwise the DNA sample is judged to be an early liver cancer negative sample.

[0163] Specifically, the following situations are included:

[0164] 1. When the first DNA molecule in the DNA sample is determined to be methylation negative, and the second DNA molecule in the DNA sample is determined to be methylation negative, the DNA sample is determined to be an early liver cancer negative sample.

[0165] 2. When the first DNA molecule in the DNA sample is determined to be methylation-positive and the second DNA molecule in the DNA sample is determined to be methylation-negative, the DNA sample is determined to be an early liver cancer-positive sample.

[0166] 3. When the first DNA molecule in the DNA sample is determined to be methylation-negative and the second DNA molecule in the DNA sample is determined to be methylation-positive, the DNA sample is determined to be an early liver cancer-positive sample.

[0167] 4. When the first DNA molecule in the DNA sample is determined to be methylation positive, and the second DNA molecule in the DNA sample is determined to be methylation positive, the DNA sample is determined to be an early liver cancer positive sample.

[0168] In addition, under the condition that the fluorescence intensity of the third fluorescent group in the PCR reaction solution reaches or exceeds the fluorescence threshold and the amplification cycle number (Ct value) of the internal reference gene is less than or equal to the third cycle threshold (such as 30 to 32), the PCR amplification is determined to be effective amplification, and the PCR amplification result of this time can be accepted. Otherwise, it is determined to be invalid amplification, and the PCR amplification result of this time cannot be accepted, and the PCR amplification needs to be repeated. After excluding the unacceptable results, the acceptable results are statistically analyzed, and the results are shown in Table 4.

[0169] In addition, because alpha fetoprotein (AFP) is a common blood marker for liver cancer detection, the detection results of AFP are used as a control experiment. The AFP concentration in the above-mentioned in vitro sample (blood sample) is detected by an alpha fetoprotein detection kit (immunochromatography). When the AFP concentration in the serum is greater than 20ng / mL, the in vitro sample is judged to be a positive sample for liver cancer detection; when the AFP concentration in the serum is less than or equal to 20ng / mL, the sample is judged to be a negative sample for liver cancer detection. The judgment results are compared with the pathological test results of the subjects, and the sensitivity and specificity of the detection using AFP are calculated. The results are shown in Table 4.

[0170] Table 4 Test results in the examples of this application

[0171]

[0172] According to the results in Table 4, when only DOK1 gene and PITX1 gene are detected, their sensitivity and specificity are higher than AFP. When DOK1 gene and PITX1 gene are detected together, higher liver cancer detection sensitivity and specificity can be achieved, and their performance is significantly better than the traditional serum protein marker AFP.

[0173] Those skilled in the art will appreciate that the methylation level of the above nucleotide sequence can be detected by a DNA methylation level detection method known in the art, such as a methylation-specific PCR method, a bisulfite treatment and sequencing method, a restriction endonuclease analysis method combined with sodium bisulfite, a methylation-specific fluorescent quantitative PCR method, a methylation-sensitive high-resolution melting curve analysis method or a pyrophosphate sequencing method. Accordingly, the reaction reagents and the detection reagents are necessary reagents in the above method. In some embodiments, the reaction reagent is selected from bisulfite or its derivatives.

[0174] In the present application, "patient" or "subject" refers to a human being diagnosed with early liver cancer for the first time. The patient can be an individual who is undergoing treatment for early liver cancer, or any individual who wishes to use the method of the present application for detection or diagnosis.

[0175] "Methylation level" represents the proportion of one or more sites in a nucleotide sequence that are in a methylated state, for example, the methylation level of a region / interval in the whole genome DNA refers to the proportion of all CpG sites in the region / interval that are in a methylated state, and the average methylation level of the whole genome DNA refers to the mean of the methylation levels of all regions / intervals in the whole genome DNA. The process of converting the results obtained by the method for detecting DNA methylation (such as simplified methylation sequencing) into methylation levels is known in the art.

[0176] The DNA in some embodiments of the present application may be single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA).

[0177] In the present application, DNA molecule A and DNA molecule B are only used to distinguish different DNA molecules, and do not mean that DNA molecule A and DNA molecule B are complementary strands of the same DNA molecule. For example, the first transformed DNA molecule A and the first transformed DNA molecule B are different DNA molecules, and the nucleotide sequences of the two are different, but they are not complementary. For example, the second transformed DNA molecule A and the second transformed DNA molecule B are different DNA molecules, and the nucleotide sequences of the two are different, but they are not complementary.

[0178] The preferred implementation methods and materials in this article are for demonstration purposes only and cannot limit the content of this application.

[0179] Unless otherwise stated, the raw materials and reagents used in the examples of the present application are commercially available or can be prepared by methods known in the art.

[0180] The kit QIAamp Circulating Nucleic Acid Kit (Qiagen) involved in the examples of the present application was purchased from Qiagen Biotech (Shanghai) Co., Ltd. The Zymo bisulfite conversion kit was purchased from Zymo. The AFP concentration in the in vitro sample (serum sample) was detected using an alpha-fetoprotein detection kit (immunochromatography). The PCR amplification kit was purchased from Novagen. The fluorescent quantitative PCR instrument was purchased from Bio-Rad.

[0181] All operations in the examples of this application were in accordance with the manufacturer's instructions.

[0182] The above is a detailed introduction to the biomarkers, kits and applications for detecting early liver cancer provided in the embodiments of the present application. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. At the same time, for technicians in this field, according to the ideas of the present application, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. Use of an isolated biomarker in the preparation of a biomarker for determining early liver cancer, the biomarker comprising: The first DNA molecule shown as SEQ ID NO.1 and / or the second DNA molecule shown as SEQ ID NO.

2.

2. Use of an isolated biomarker in the preparation of a test object for a kit for determining whether an ex vivo sample is an early liver cancer positive sample, the biomarker comprising: A first DNA molecule as shown in SEQ ID NO.1 and / or a second DNA molecule as shown in SEQ ID NO.2; The first DNA molecule is selected from the DOK1 gene with genomic coordinates chr2:74555005-74555272; and / or The second DNA molecule is selected from the PITX1 gene with genomic coordinates of chr5:135027454-135027645.

3. A kit for identifying positive samples of early liver cancer, characterized in that: The kit comprises: A conversion reagent is provided to convert unmethylated cytosine in a first DNA molecule containing a methylated CpG site in a cfDNA sample into uracil to obtain a first converted DNA molecule A, and convert all cytosine in the first DNA molecule without a methylated CpG site in the cfDNA sample into uracil to obtain a first converted DNA molecule B; the nucleotide sequence of the first DNA molecule is shown in SEQ ID NO.1; and / or, convert unmethylated cytosine in a second DNA molecule containing a methylated CpG site in the cfDNA sample into uracil to obtain a second converted DNA molecule A, and convert all cytosine in the second DNA molecule without a methylated CpG site in the cfDNA sample into uracil to obtain a second converted DNA molecule B, and the nucleotide sequence of the second DNA molecule is shown in SEQ ID NO.2; A first primer pair is used for PCR amplification of the first converted DNA molecule A, and a first fluorescent probe, including a first fluorescent group, a first DNA probe and a first quenching group connected in sequence; and / or a second primer pair is used for PCR amplification of the second converted DNA molecule A, and a second fluorescent probe, including a second fluorescent group, a second DNA probe and a second quenching group connected in sequence; Wherein, the PCR is methylation-specific fluorescent quantitative PCR.

4. The kit according to claim 3, characterized in that The first fluorescent group and the second fluorescent group are each independently selected from FAM, Cy5, VIC, TET, HEX, JOE, ROX; and / or The first fluorescent group and the second fluorescent group are different types of fluorescent groups; and / or The cfDNA sample is from blood, serum or plasma; and / or The first primer pair includes a first forward primer as shown in SEQ ID NO.3 and a first reverse primer as shown in SEQ ID NO.4; and / or The second primer pair comprises a second forward primer as shown in SEQ ID NO.5 and a second reverse primer as shown in SEQ ID NO.6; and / or The nucleotide sequence of the first DNA probe is shown in SEQ ID NO.7; and / or The nucleotide sequence of the second DNA probe is shown as SEQ ID NO.

8.

5. The kit according to claim 3, characterized in that The kit also includes: A third primer pair, used for PCR amplification of an internal reference gene in the cfDNA sample; and The third fluorescent probe includes a third fluorescent group, a third DNA probe and a third quenching group which are connected in sequence.

6. The kit according to claim 5, characterized in that The internal reference gene is selected from any one of the ACTB gene, RNase P gene, GAPDH gene, TBP gene and HPRT gene; and / or The third primer pair includes a third forward primer as shown in SEQ ID NO.9 and a third reverse primer as shown in SEQ ID NO.10; and / or The nucleotide sequence of the third DNA probe is shown in SEQ ID NO.11; and / or The third fluorescent group is selected from FAM, Cy5, VIC, TET, HEX, JOE, ROX; and / or The third fluorescent group, the first fluorescent group and the second fluorescent group are fluorescent groups of different types.

7. The kit according to claim 3, characterized in that The kit further comprises: an extraction reagent for extracting the cfDNA sample from an in vitro sample; and / or The conversion reagent comprises bisulfite; and / or PCR amplification reagent, using the first primer pair and the first fluorescent probe to perform PCR amplification on the first converted DNA molecule A; and / or using the second primer pair and the second fluorescent probe to perform PCR amplification on the second converted DNA molecule A.

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