CN118048451A - Marker for screening liver cancer, probe composition and application thereof - Google Patents

Marker for screening liver cancer, probe composition and application thereof Download PDF

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Publication number
CN118048451A
CN118048451A CN202211442630.5A CN202211442630A CN118048451A CN 118048451 A CN118048451 A CN 118048451A CN 202211442630 A CN202211442630 A CN 202211442630A CN 118048451 A CN118048451 A CN 118048451A
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marker
seq
probe
composition
methylation
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Inventor
吴宁宁
韩晓亮
关晋霞
王秀秀
连明
郭媛媛
魏闯
彭勇飞
王小奇
田继超
杨亚东
李永君
周光朋
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Biochain Beijing Science and Technology Inc
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Biochain Beijing Science and Technology Inc
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Abstract

The application discloses a marker for screening liver cancer, a probe composition and application thereof. The marker can sensitively and specifically detect the methylation state of the gene, so that the marker can be used for detecting free DNA of peripheral blood, and the composition is used for screening asymptomatic people in a non-invasive mode, reduces the harm caused by invasive detection, has higher sensitivity and accuracy, and can realize real-time monitoring.

Description

Marker for screening liver cancer, probe composition and application thereof
Technical Field
The application relates to the technical field of biology, in particular to a methylation detection marker for screening liver cancer, a probe composition and application thereof.
Background
The main reason for low survival rate of liver cancer is that screening of high risk group of liver cancer is not popular, early diagnosis rate is low, and 70% -80% of patients are middle and late stage in diagnosis, if early detection and early diagnosis can be carried out, radical means such as liver resection and liver transplantation can be implemented, and prognosis of liver cancer patients can be obviously improved; and secondly, the recurrence and transfer rate reaches 40-70% after 5 years of liver cancer excision operation. The existing diagnosis and treatment strategies and measures are very limited in reducing the total illness and death rate of liver cancer for 5 years, so that the search for new liver cancer screening and diagnosis and treatment strategies is urgent. Currently, DNA methylation has been demonstrated to be tissue specific, useful in early cancer detection, and can be traced to the primary tumor site based on the methylation profile of circulating tumor DNA (ctDNA).
Disclosure of Invention
The application provides a marker for detecting liver cancer, which can be used for screening liver cancer, is used for screening asymptomatic people in a non-invasive mode and detecting prognosis of cancer patients, reduces the damage caused by invasive detection, has higher sensitivity and accuracy, and can realize implementation monitoring. The application also provides a composition and a kit for detecting liver cancer.
In particular, the application adopts the following technical scheme,
1. A marker for detecting liver cancer, the marker being selected from one or more of the following genes: PSD4, MIR4783, PLAC8, TSTYL 5, FAR1, PPFIA1, KIF7.
2. The marker according to item 1, wherein the nucleotide sequence of the marker is selected from the group consisting of SEQ ID NOs: 1-SEQ ID NO:12, preferably the marker is a methylated marker.
3. A probe composition for detecting liver cancer, the probe composition comprising a nucleic acid that targets methylation of the marker of item 1 or 2.
4. The probe composition of item 3, wherein the probe composition comprises a hypermethylated first probe composition for hybridization with a bisulfite converted CG hypermethylated region and a hypomethylated second probe composition for hybridization with a bisulfite converted CG hypomethylated region;
preferably, the first probe composition comprises n probes that hybridize to each nucleotide of the sense strand and/or the antisense strand of the bisulfite-converted CG hypermethylated region;
preferably, the second probe composition comprises m probes that hybridize to each nucleotide of the sense strand and/or the antisense strand of the bisulfite-converted CG hypomethylated region;
preferably, n and m are each any integer from 1 to 10;
Preferably, there is an overlap of x 1 nucleotides between the n-1 th probe and the n-th probe, preferably x 1 is any integer from 0 to 100;
Preferably, there is an overlap of x 2 nucleotides between the m-1 th probe and the m-th probe, preferably x 2 is any integer from 0 to 100;
further preferred, the first probe composition comprises a nucleotide sequence as set forth in SEQ ID NO:61-88, or two or four or six or eight or ten or twelve of said second probe compositions comprising the amino acid sequence as set forth in SEQ ID NO:89-116, two or four or six or eight or ten or twelve.
5. Use of a nucleic acid molecule for detecting a marker selected from one or more of the following genes in the preparation of a kit for detecting liver cancer: PSD4, MIR4783, PLAC8, TSTYL 5, FAR1, PPFIA1, KIF7.
6. The use according to item 5, wherein the nucleotide sequence of the target sequence of the marker is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 1-9, preferably the marker is a methylated marker;
preferably, the probe composition is used for targeting a methylated marker of liver cancer;
Preferably, the probe composition is the probe composition according to item 3 or 4.
7. A composition for liver cancer detection, the composition comprising a nucleic acid for detecting a methylation state of any one or more markers selected from the group consisting of: PSD4, MIR4783, PLAC8, TSTYL 5, FAR1, PPFIA1, KIF7.
8. The composition of item 7, wherein the nucleotide sequence of the marker is selected from the group consisting of SEQ ID NOs: 1-SEQ ID NO: 12.
9. The composition of item 7 or 8, wherein the nucleic acid comprises the probe composition of item 3 or 4;
Preferably, the nucleic acid comprises:
A primer that is a fragment of at least 9 nucleotides in the marker, the fragment comprising at least one CpG dinucleotide sequence;
Preferably, the nucleic acid further comprises:
A probe that hybridizes under moderately stringent or stringent conditions to at least 15 nucleotide fragments in said marker, said fragments comprising at least-CpG dinucleotide sequences;
Preferably, the composition further comprises an agent that converts the unmethylated cytosine base at position 5 of the marker to uracil;
Preferably, the nucleic acid for detecting methylation of a marker further comprises:
Blocking agents that preferentially bind to target sequences in the unmethylated state.
10. A kit comprising reagents for detecting a marker according to item 1 or 2 or a probe composition according to item 3 or 4 or a composition according to any one of items 7 to 9.
11. A chip comprising a reagent for detecting the marker of item 1 or 2 or the probe composition of item 3 or 4 or the composition of item 7 or 8.
Effects of the invention
The inventor of the application utilizes the epigenomic and bioinformatics technology, finds out a plurality of methylation genes related to liver cancer by analyzing genome methylation data of the liver cancer, determines a target sequence of methylation abnormality of the methylation genes of the liver cancer, and can sensitively and specifically detect the methylation state of the genes by the target sequence of the methylation genes, thereby being used for detecting free DNA of peripheral blood.
The composition is used for screening asymptomatic people in a non-invasive mode, reduces the harm caused by invasive detection, has higher sensitivity and accuracy, and can realize real-time monitoring.
Detailed Description
Exemplary embodiments of the application are described below, including various details of embodiments of the application to facilitate understanding, which should be considered as merely exemplary. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the application. Also, descriptions of well-known functions and constructions are omitted in the following description for clarity and conciseness.
The application provides a marker for detecting liver cancer, which is selected from one or more than two of the following genes: PSD4, MIR4783, PLAC8, TSTYL 5, FAR1, PPFIA1, KIF7.
In one embodiment the nucleotide sequence of the marker is selected from the group consisting of SEQ ID NO:1-SEQ ID NO:12, preferably, the marker is a methylated marker.
Wherein, the nucleotide sequence of PSD4 is shown in SEQ ID NO:1 and SEQ ID NO: shown as 9; the nucleotide sequence of MIR4783 is shown in SEQ ID NO:2 and SEQ ID NO:3 is shown in the figure; the nucleotide sequence of PLAC8 is shown in SEQ ID NO:4 and SEQ ID NO:10 is shown in the figure; the nucleotide sequence of TSPYL5 is shown in SEQ ID NO:5 is shown in the figure; the nucleotide sequences of FAR1 are shown in SEQ ID NO:6 and SEQ ID NO: 11; the nucleotide sequences of PPFIA1 are respectively shown in SEQ ID NO:7 and SEQ ID NO: shown at 12; the nucleotide sequence of KIF7 is shown in SEQ ID NO: shown at 8.
Wherein the sequences of the markers are all sequences which are not converted by bisulfite.
The present invention provides a probe composition comprising probes that target the different degrees of methylation state of the marker (after bisulfite conversion).
Methylation refers to methylation of the 5 th carbon atom on cytosine in CpG dinucleotides, and is taken as a stable modification state, and can inherit new generation progeny DNA along with the DNA replication process under the action of DNA methyltransferase, so that the methylation is an important epigenetic mechanism, and DNA methylation of a gene promoter region can lead to silence of transcription of cancer suppressor genes, so that the methylation has a close relation with tumor occurrence. Aberrant methylation includes hypermethylation of cancer suppressor genes and DNA repair genes, hypomethylation of repeated sequence DNA, imprinting loss of certain genes, which are associated with the occurrence of a variety of tumors.
Methylation according to the present application may be methylation level, degree of methylation or methylation status, and when analyzing methylation of such target sequences, a person skilled in the art may use quantitative determination methods to determine methylation.
The probe is single-stranded or double-stranded DNA with a length of tens to hundreds or even thousands of base pairs, which can utilize the denaturation, renaturation and high precision of base complementary pairing of molecules to form double-stranded complex (hybrid) by hydrogen bonding (hybridization) with complementary unlabeled single-stranded DNA or RNA in a sample to be tested. After washing off the unpaired and bound probe, the hybridization reaction results can be detected by a detection system such as an autoradiography or an enzyme-linked reaction. In the present application, the region that complementarily binds or hybridizes to the probe is a specific target region, and a plurality of probes are combined into a probe composition.
In one embodiment, the probe composition comprises a first probe composition for targeting the high methylation state of the marker (after bisulfite conversion) for hybridization with the high methylation marker converted by bisulfite or its complement and a second probe composition for targeting the low methylation state of the marker (after bisulfite conversion) for hybridization with the low methylation marker converted by bisulfite or its complement.
In the present application, hypermethylation means that after the marker is converted by bisulfite, base C becomes base U, but if it is base CG, base C remains unchanged;
In the present application, the hypomethylation means that after the marker is converted by bisulfite, all bases CG are not methylated, and the bases C are changed into the bases U.
Since the methylation status varies from person to person, the sequence of the tag converted by bisulfite varies, one extreme case of the tag is shown here, i.e. all CG of the segment is in methylated hypermethylation status, and the hypermethylation status sequence of its complementary strand: SEQ ID NO:1 is set forth in SEQ ID NO: 13; SEQ ID NO:1 in the complementary strand, the sequence of one extreme of the hypermethylation state of the complementary strand is set forth in SEQ ID NO: 14; SEQ ID NO:2 is set forth in SEQ ID NO: 15; SEQ ID NO:2 of the complementary strand of SEQ ID NO: shown at 16; SEQ ID NO:3 is set forth in SEQ ID NO: shown at 17; SEQ ID NO:3 of the complementary strand, the sequence of one extreme of the hypermethylation state of the complementary strand is set forth in SEQ ID NO: shown at 18; SEQ ID NO:4 is set forth in SEQ ID NO: 19; SEQ ID NO:4 of the complementary strand of SEQ ID NO: shown at 20; SEQ ID NO:5 is set forth in SEQ ID NO: 21; SEQ ID NO:5, the sequence of one extreme of the hypermethylation state of the complementary strand is set forth in SEQ ID NO:22, as shown in: SEQ ID NO:6 is set forth in SEQ ID NO: indicated at 23; SEQ ID NO:6 complementary strand hypermethylation state of the sequence set forth in one extreme of SEQ ID NO: shown at 24; SEQ ID NO:7 is set forth in SEQ ID NO: shown at 25; SEQ ID NO:7 of complementary strand of SEQ ID NO: 26; SEQ ID NO:8 is set forth in SEQ ID NO: shown at 27; SEQ ID NO:8 complementary strand of SEQ ID NO: 28; SEQ ID NO:9 is set forth in SEQ ID NO: 29; SEQ ID NO:9 of the complementary strand, the sequence of one extreme of the hypermethylation state of the complementary strand is set forth in SEQ ID NO: shown at 30; SEQ ID NO:10 is set forth in SEQ ID NO: 31; SEQ ID NO:10 of the complementary strand is set forth in SEQ ID NO: shown at 32; SEQ ID NO:11 is set forth in SEQ ID NO:33, as shown in: SEQ ID NO:11 of the complementary strand is set forth in SEQ ID NO: shown at 34; SEQ ID NO:12 is set forth in SEQ ID NO: indicated at 35; SEQ ID NO: the sequence of one extreme case of hypermethylation state of the 12 complementary strand is set forth in SEQ ID NO: shown at 36;
Similarly, because of the different methylation states of each individual, an extreme case is shown where all CG is in an unmethylated hypomethylated state, as is the sequence of hypomethylation states of their complementary strands: SEQ ID NO:1 is set forth in SEQ ID NO: shown at 37; SEQ ID NO:1, the sequence of one extreme of the hypomethylation state of the complementary strand is set forth in SEQ ID NO: shown at 38; SEQ ID NO:2 is set forth in SEQ ID NO: 39; SEQ ID NO:2 of the complementary strand of SEQ ID NO: shown at 40; SEQ ID NO:3 is set forth in SEQ ID NO: 41; SEQ ID NO:3 complementary strand has the sequence set forth in SEQ ID NO: 42; SEQ ID NO:4 is set forth in SEQ ID NO: 43. SEQ ID NO:4 of complementary strand of SEQ ID NO: shown at 44; SEQ ID NO:5 is set forth in SEQ ID NO: 45; SEQ ID NO:5 complementary strand has the sequence set forth in SEQ ID NO: 46; SEQ ID NO:6 is set forth in SEQ ID NO: indicated at 47; SEQ ID NO:6 complementary strand has the sequence set forth in SEQ ID NO: 48; SEQ ID NO:7 is set forth in SEQ ID NO: shown at 49; SEQ ID NO:7 complementary strand has the sequence set forth in SEQ ID NO: shown at 50; SEQ ID NO:8 is set forth in SEQ ID NO: 51; SEQ ID NO:8 complementary strand has the sequence set forth in SEQ ID NO: 52; SEQ ID NO:9 is set forth in SEQ ID NO: 53; SEQ ID NO:9 complementary strand has the sequence set forth in SEQ ID NO: indicated at 54; SEQ ID NO:10 is set forth in SEQ ID NO: indicated at 55; SEQ ID NO:10 of the complementary strand is set forth in SEQ ID NO: shown at 56; SEQ ID NO:11 is set forth in SEQ ID NO: 57; SEQ ID NO:11 is set forth in SEQ ID NO: indicated at 58; SEQ ID NO:12 is set forth in SEQ ID NO: 59; SEQ ID NO: the sequence of one extreme of the hypomethylation state of the 12 complementary strand is set forth in SEQ ID NO: shown at 60.
In one embodiment, the first probe composition comprises n probes that hybridize to each nucleotide of the hypermethylated sense and/or antisense strand region of the bisulfite converted marker.
The second probe composition comprises m probes that hybridize to each nucleotide of the hypomethylated sense and/or antisense strand region of the bisulfite converted marker.
The number of probes in the first probe composition and the second probe composition is not limited in any way, and those skilled in the art can select the number as desired, for example, m and n may be any integer of 1 to 10, and m and n may be the same or different.
For example, m and n may be any integer of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, preferably, m=n=2.
In one embodiment, there is an overlap of x1 nucleotides between the n-1 th probe and the n-th probe, preferably x 1 is any integer from 0 to 100;
preferably, there is an overlap of x 2 nucleotides between the m-1 th probe and the m-th probe, and preferably x 2 is any integer from 0 to 100.
Wherein x 1 and x 2 may be the same or different, and when x 1 is 0, the tail of the n-1 probe is connected to the head of the n-th probe, and similarly, when x 2 is 0, the tail of the m-1 probe is connected to the head of the m-th probe.
According to the application, the probe composition is hybridized with the marker converted by the bisulfite or the complementary sequence converted by the bisulfite, wherein the first probe composition of the target high-methylation marker is hybridized with the high-methylation marker region, and the second probe composition of the target low-methylation marker is hybridized with the low-methylation marker region, so that the methylation level of the target sequence can be detected efficiently and accurately, and the target sequence can be further used for liver cancer screening.
In one embodiment, the first probe composition targeting the hypermethylated said marker comprises a sequence as set forth in SEQ ID NO:61-88, or two or four or six or eight or ten or twelve.
The second probe composition targeting the hypomethylated marker comprises a nucleotide sequence as set forth in SEQ ID NO:89-116, two or four or six or eight or ten or twelve.
Wherein the first probe composition for hybridization to a PSD4 methylated sequence comprises a nucleotide sequence as set forth in SEQ ID NO:61-62 (for hybridization with the methylated sequence of SEQ ID NO: 1) and the nucleotide sequence set forth in SEQ ID NO:81-82 (for hybridization with the methylated sequence of SEQ ID NO: 9);
A first probe composition for hybridization to MIR4783 methylation sequences comprises the sequence set forth in SEQ ID NO:63-64 (for hybridization with the methylation sequence of SEQ ID NO: 2) and the nucleotide sequence set forth in SEQ ID NO:65-66 (for hybridization with the methylated sequence of SEQ ID NO: 3);
a first probe composition for hybridization to a PLAC8 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NO:67-68 (for hybridization with the methylation sequence of SEQ ID NO: 4) and the nucleotide sequence set forth in SEQ ID NO:83-84 (for hybridization with the methylated sequence of SEQ ID NO: 10);
A first probe composition for hybridization to a TSPYL5 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NO: 69-70;
A first probe composition for hybridization to FAR1 methylation sequences comprises a nucleotide sequence as set forth in SEQ ID NO:71-72 (for hybridization with the methylated sequence of SEQ ID NO: 6) and the nucleotide sequence set forth in SEQ ID NO:85-86 (for hybridization with the methylated sequence of SEQ ID NO: 11);
A first probe composition for hybridization to PPFIA1 methylation sequences comprises a nucleotide sequence as set forth in SEQ ID NO: 73-74;
a first probe composition for hybridization to KIF7 methylation sequences comprises a nucleotide sequence as set forth in SEQ ID NO: 77-80;
A second probe composition for hybridization to a PSD4 methylated sequence comprises a nucleotide sequence as set forth in SEQ ID NO:89-90 (for hybridization with the methylated sequence of SEQ ID NO: 1) and the nucleotide sequence set forth in SEQ ID NO:109-110 (for hybridization with the methylated sequence of SEQ ID NO: 9);
A second probe composition for hybridization to MIR4783 methylation sequences comprises the sequence set forth in SEQ ID NO:91-92 (for hybridization with the methylation sequence of SEQ ID NO: 2) and the nucleotide sequence set forth in SEQ ID NO:93-94 (for hybridization with the methylated sequence of SEQ ID NO: 3);
A second probe composition for hybridization to a PLAC8 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NO:95-96 (for hybridization with the methylation sequence of SEQ ID NO: 4) and the nucleotide sequence shown in SEQ ID NO:112-113 (for hybridization with the methylated sequence of SEQ ID NO: 10);
A second probe composition for hybridization to a TSPYL5 methylated sequence comprises a sequence as set forth in SEQ ID NO: 97-98;
A second probe composition for hybridization to FAR1 methylation sequences comprises a nucleotide sequence as set forth in SEQ ID NO:99-100 (for hybridization with the methylated sequence of SEQ ID NO: 6) and the nucleotide sequence set forth in SEQ ID NO:85-86 (for hybridization with the methylated sequence of SEQ ID NO: 11);
a second probe composition for hybridization to PPFIA1 methylation sequences comprises a nucleotide sequence as set forth in SEQ ID NO: 103-104;
A second probe composition for hybridization to KIF7 methylation sequences comprises a nucleotide sequence as set forth in SEQ ID NO: 105-108.
The application provides application of the marker in preparation of a kit for detecting liver cancer, wherein the marker is selected from one or more than two of the following genes: PSD4, MIR4783, PLAC8, TSTYL 5, FAR1, PPFIA1, KIF7.
In one embodiment, the nucleotide sequence of the marker is selected from the group consisting of SEQ ID NOs: 1-SEQ ID NO:12, preferably the marker is a methylated marker.
The application provides application of a nucleic acid molecule for detecting the marker in preparation of a kit for detecting liver cancer, and the probe composition is used for targeting the marker after methylation of the liver cancer.
In one embodiment, the nucleic acid molecule is a composition of probes as described above.
The application provides a composition for liver cancer detection, which comprises nucleic acid for detecting any one or more than two methylation markers selected from the following markers: PSD4, MIR4783, PLAC8, TSTYL 5, FAR1, PPFIA1, KIF7. Preferably, the nucleotide sequence of the marker is selected from the group consisting of SEQ ID NOs: 1-SEQ ID NO: 12.
In one embodiment, the nucleic acid comprises a probe composition as described above.
In one embodiment, the nucleic acid comprises:
a primer that is a fragment of at least 9 nucleotides in the marker, the fragment comprising at least one CpG dinucleotide sequence.
Wherein, if bisulfite is used to convert the sample DNA to be tested, the nucleic acid for detecting methylation of the marker comprises a fragment of at least 9 nucleotides in the sequence after bisulfite conversion of the target sequence of the marker, said fragment comprising at least one CpG dinucleotide sequence.
In one embodiment, the nucleic acid further comprises:
a probe that hybridizes under moderately stringent or stringent conditions to at least 15 nucleotide fragments in said marker, said fragments comprising at least one CpG dinucleotide sequence.
In one embodiment, the composition further comprises an agent that converts the unmethylated cytosine base at the 5-position of the marker to uracil, e.g., the agent can be bisulfite or the like; preferably, the nucleic acid for detecting methylation of a marker further comprises:
Blocking agents that preferentially bind to target sequences in the unmethylated state.
The blocker is used for improving the amplification specificity of the PCR amplification primer, the 5 '-end of the blocker nucleotide sequence and the 3' -end nucleotide sequence of the forward or reverse primer have an overlapping region of more than or equal to 5 nucleotides, the blocker is complementary with the forward or reverse primer and the same strand of target gene target sequence DNA, the melting temperature of the blocker is higher than that of the forward or reverse primer by more than (including) 5 ℃, and the nucleotide sequence of the blocker comprises at least one CpG dinucleotide sequence and is complementary with the sequence of the target gene target sequence DNA which is not subjected to methylation after the conversion of the bisulfite. Thus, when the genomic DNA of the biological sample to be detected is a mixture of methylated and unmethylated state, especially in the case where the DNA in the methylated state is far less than the DNA in the unmethylated state, the DNA in the unmethylated state is converted by bisulfite and then preferentially binds to the blocker, thereby inhibiting the binding of the DNA template to the PCR primer and thus not causing PCR amplification, whereas the DNA in the methylated state does not bind to the blocker and thus does not bind to the primer set, PCR amplification occurs, followed by direct or indirect detection of the fragment obtained by the amplification.
The application provides a kit comprising reagents for detecting the above markers or the above probe composition or the above composition.
In one embodiment, the kit further comprises a container for holding a biological sample of a subject.
In one embodiment, the kit further comprises instructions for use and interpretation of the test results.
The biological sample may be, for example, peripheral blood whole blood, plasma or serum.
The present invention is not limited in any way to a method for detecting methylation level of a target sequence using the above-described kit, and one skilled in the art can select according to need, for example, the present invention provides a method for detecting methylation level of a target sequence of a marker using the above-described kit, comprising the steps of:
Collecting a sample of a subject; extracting and purifying DNA in the sample; constructing a DNA library for sequencing against the purified DNA sample; transforming said constructed DNA library with bisulfite; pre-PCR amplifying the bisulfite converted DNA library; performing hybridization capture on the sample amplified by the pre-PCR by using the probe composition; amplifying the hybridized and captured product by utilizing PCR; performing high-throughput second-generation sequencing on the PCR amplified product after hybridization capture; analyzing the sequencing data to determine the methylation level of the sample; calculating a threshold value for each marker based on methylation of an existing sample, interpreting the patient's disease based on the methylation level of a certain marker of the sample, if the methylation level of a certain marker of the sample exceeds the threshold value, it is a cancer sample, if it is below the threshold value, it is a healthy human sample.
Also for example, the present invention provides a method for detecting the methylation level of a marker using the above-described kit, comprising the steps of: (1) withdrawing peripheral blood from the subject, and separating plasma or serum; (2) extracting free DNA from plasma or serum; (3) Treating the free DNA from step (2) with a reagent to convert the unmethylated cytosine base at position 5 to uracil or other bases, i.e., converting the unmethylated cytosine base at position 5 of the marker to uracil or other bases, the converted bases differing from the unmethylated cytosine base at position 5 in hybridization performance and being detectable; (4) Contacting the free DNA treated in step (3) with a DNA polymerase and primers for the markers such that the treated markers are amplified to produce amplified products or are not amplified; the treated marker, if subjected to DNA polymerization, produces amplification products; the treated marker is not amplified if DNA polymerization does not occur; (5) detecting the amplified product with a probe; (6) Determining the methylation status of at least one CpG dinucleotide of the marker based on the presence or absence of the amplification product, thereby determining the methylation level of the marker.
The invention provides a chip comprising a reagent for detecting the marker described above or the probe composition described above or the composition described above.
The agent may be a nucleic acid molecule.
The sequencing principle of the chip, also called a gene chip, is a hybridization sequencing method, namely a method for determining the sequence of nucleic acid by hybridizing with a group of nucleic acid probes with known sequences, wherein probes with target nucleotides with known sequences are immobilized on the surface of a substrate. When the nucleic acid sequence with fluorescent mark in the solution is complementarily matched with the nucleic acid probe at the corresponding position on the gene chip, a group of probe sequences with complete complementation of the sequences are obtained by determining the probe position with the strongest fluorescence intensity.
The chip is prepared by mainly taking a glass sheet or a silicon wafer as a carrier, and sequentially arranging oligonucleotide fragments or cDNA (complementary deoxyribonucleic acid) serving as probes on the carrier by adopting an in-situ synthesis and microarray method.
The chip is based on signal detection of DNA sequence hybridization after bisulfite treatment, wherein unmethylated cytosine is changed into uracil, methylated cytosine is kept unchanged, uracil is converted into thymine, and finally chip hybridization is carried out; finally, judging the type of the added base according to the fluorescence color, and further determining whether the locus is methylated.
The application provides a liver cancer screening method, which comprises the following steps:
detecting the methylation level of the marker, and
Determining the risk of the subject for developing liver cancer based on the methylation level, wherein the marker is selected from one or more of the following:
PSD4、MIR4783、PLAC8、TSPYL5、FAR1、PPFIA1、KIF7。
Examples
The materials used in the test and the test methods are described generally and/or specifically in the examples which follow,% represents wt%, i.e. weight percent, unless otherwise specified. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
Example 1 screening markers
1) Sample collection: the 450k methylated chip cancer tissue data in TCGA was downloaded, and included in 7769 cancer tissue samples from 26 tumors, including adrenocortical carcinoma (80), bladder urothelial carcinoma (409), acute myeloid leukemia (140), brain low grade glioma (654), breast carcinoma (740), cervical carcinoma (286), colorectal carcinoma (348), esophageal carcinoma (183), uveal melanoma (80), head and neck squamous cell carcinoma (527), renal carcinoma (660), liver carcinoma (377), lung adenocarcinoma (425), lung squamous carcinoma (372), diffuse large B-cell lymphoma (29), ovarian serous cyst adenocarcinoma (10), pancreatic carcinoma (184), mesothelioma (116), prostate carcinoma (488), skin melanoma (104), sarcoma (117), gastric carcinoma (397), testicular carcinoma (134), thymus carcinoma (94), thyroid carcinoma (506), endometrial carcinoma (309). For healthy people, the blood plasma of 38 healthy people was collected in the Bohr's way, and genome-wide methylation sequencing was performed (Who 1e Genome Bisulfite Sequencing, WGBS).
2) Candidate marker screening: 450K data were processed using an open source R-pack (CHAMP), and healthy plasma samples were differentially aligned with each cancer tissue, with P < 0.05 defined as the differential interval. Then find the interval of difference only in liver cancer, combine the dual significance of statistics and functional, have chosen 2000 markers to carry on the study below.
3) And (3) marker selection: of the 2000 markers, the difference in methylation level between 450 k-chip liver cancer tissue (377) and paracancerous tissue (53) in TCGA was required to be greater than 0.2, and the first 50 differential methylation regions were selected according to the order of differential size. Since the distribution of DNA methylation rates over the genome tends to exhibit the characteristic of "more adjacent, more similar", many studies have shown that methylation rates within 2kb of each other over the genome exhibit a clear correlation, i.e., co-methylation. Thus, for the 50 markers described above, 1kb was extended upstream and downstream.
4) And (3) marker verification: the 50 different methylation areas are designed for probe capture, and the data of Bohr's plasma samples (the number of liver cancer samples=40 and the number of healthy human samples=40) are used for verification, so that 12 markers capable of distinguishing liver cancer from healthy people are finally obtained. The sequences of which are SEQ ID NOs: 1-SEQ ID NO: shown at 12.
5) Custom panel verification: based on the resulting target sequence region, a probe composition (panel) is tailored.
Then, the sample is verified in a plasma sample, and the experimental detection method is as follows
Cfdna extraction purification
1.1.1. Plasma sample preparation:
The blood samples were centrifuged at 2000g for 10min at 4℃and the plasma was transferred to a new centrifuge tube. The plasma samples were centrifuged at 16000g for 10min at 4℃and the next step was performed depending on the type of collection tube used, which was other in this experiment, as shown in Table 1.
TABLE 1
1.1.2. Cleavage and binding
1.1.2.1. The binding solution/bead mixture was prepared according to table 2 below and then thoroughly mixed.
TABLE 2
An appropriate volume of plasma sample was added.
1.1.2.2. The plasma sample and binding solution/bead mixture are thoroughly mixed.
1.1.2.3. The cfDNA was bound to the magnetic beads by sufficient binding on a spin mixer for 10 min.
1.1.2.4. The binding tube was placed on a magnetic rack for 5min until the solution became clear and the beads were fully adsorbed on the magnetic rack.
1.1.2.5. The supernatant was carefully discarded with a pipette, the tube was kept on the magnetic rack for several minutes, and the residual supernatant was removed with a pipette.
1.1.3. Washing
1.1_3.1. The beads were resuspended in 1ml of wash solution.
1.1.3.2. The resuspension was transferred to a new non-adsorbed 1.5ml centrifuge tube. The binding tube remains.
1.1_3.3. Centrifuge tubes containing the bead resuspension were placed on a magnet rack for 20s.
1.1_3.4. The supernatant from the separation was aspirated and the binding tube was washed, the washed residual beads were again collected in a resuspension and the lysis/binding tube discarded.
1.1.3.5. The tube was placed on a magnet rack for 2min until the solution became clear, the beads were collected on the magnet rack and the supernatant was removed with a 1ml pipette.
1.1.3.6. The tube was left on the magnet rack and the remaining liquid was removed as much as possible with a 200. Mu.L pipette.
1.1.3.7. The tube was removed from the magnet holder, 1ml of wash solution was added and vortexed for 30s.
1.1.3.8. The solution was allowed to settle for 2min on a magnetic rack, the beads were collected on the magnetic rack, and the supernatant was removed with a 1ml pipette.
1.1.3.9. The tube was left on the magnet rack and the residual liquid was removed thoroughly with a 200 μl pipette.
1.1.3.10. The tube was removed from the magnet holder, 1ml 80% ethanol was added, and vortexed for 30s.
1.1.3.11. The solution was allowed to settle for 2min on a magnetic rack and the supernatant was removed with a 1ml pipette.
1.1.3.12. The tube was left on the magnet holder and the residual liquid was removed with a 200. Mu.L pipette.
1.1.3.13. The above 1.1.3.10.— 1.1.3.12 steps were repeated with 80% ethanol once, and the supernatant was removed as much as possible.
1.1.3.14. The tube was left on the magnetic rack and the beads were dried in air for 3-5 minutes.
1.1.4. Elution of cfDNA
1.1.4.1. The eluent was added as in table 3 below.
TABLE 3 Table 3
1.1.4.2. Vortex for 5min, place on a magnetic rack for 2min, the solution becomes clear, and suck cfDNA in the supernatant.
1.1.4.3. The purified cfDNA was used immediately or the supernatant was transferred to a new centrifuge tube and stored at-20 ℃.
Disruption and purification of gDNA:
1.2.1. according to the Qubit concentration, 2. Mu.g gDNA was taken, added with water to 125. Mu.l, added to covaris. Mu.l of the break tube, and the procedure was set: 50W,20%,200 cycles, 250s.
1.2.2. After the interruption, 1 μl of the sample is taken and subjected to fragment detection by using Agilent2100, and after normal interruption, the main peak of the sample detection is about 150bp-200bp.
For cfDNA samples, agilent2100 performed fragment detection, and direct Qubit was used for subsequent experiments.
1.3. Terminal repair, 3' end plus "a":
1.3.1. 20ng of the cut gDNA or cfDNA was added to a PCR tube, and 50. Mu.l of the mixture was supplemented with nuclease-free water, and the reagents in Table 4 were added and vortexed to mix well:
TABLE 4 Table 4
Component (A) Volume of
gDNA/cfDNA 50μl
Stop repair and A tailing buffer 7μl
Termination repair and A tailing enzyme mixture 3μl
Total volume of 60μl
1.3.2. The following procedure in table 5 was set up to perform the reaction on a PCR instrument: the temperature of the hot cover is 85 ℃.
TABLE 5
Temperature (temperature) Time of
20℃ 30min
65℃ 30min
4℃ ∞
1.4. Joint connection and purification:
1.4.1. the linker was diluted in advance to the appropriate concentration with reference to table 6 below:
TABLE 6
1.4.2. The reagents in the following table 7 were prepared as follows, gently swirled and mixed, and briefly centrifuged:
TABLE 7
Component (A) Volume of
End repair, addition of "A" reaction product 60μl
Joint 5μl
Nuclease-free water 5μl
Connection buffer solution 30μl
DNA ligase 10μl
Total volume of 110μl
1.4.3. The following procedure in table 8 was set up to perform the reaction on a PCR instrument: there is no thermal cover.
TABLE 8
Temperature (temperature) Time of
20℃ 30min
4℃ ∞
1.4.4. According to the system in the following table 9, purified magnetic beads are added for experiment (Agencourt AMPure XP magnetic beads are taken to room temperature in advance, and are vibrated and mixed uniformly for standby):
TABLE 9
Component (A) Volume of
Joint connection product 110μl
Agencourt AMPure XP bead 110μl
Total volume of 220μl
1.4.4.1. Gently sucking and beating, and mixing for 6 times.
1.4.4.2. Standing at room temperature for 5-15min, and placing the PCR tube on a magnetic rack for 3min to clarify the solution.
1.4.4_3. Remove supernatant, place the PCR tube on a magnetic rack, add 200. Mu.l 80% ethanol solution into the PCR tube, and stand for 30s.
1.4.4.4. The supernatant was removed, 200. Mu.l of 80% ethanol solution was added to the PCR tube, and after standing for 30s, the supernatant was thoroughly removed (it was recommended to remove the bottom residual ethanol solution using a 10. Mu.l pipette).
1.4.4.5. Standing at room temperature for 3-5min to volatilize residual ethanol thoroughly.
1.4.4.6. Adding 22 μl of nuclease-free water, removing the PCR tube from the magnetic rack, gently sucking and beating the resuspended magnetic beads, avoiding generating bubbles, and standing at room temperature for 2min.
1.4.4.7. The PCR tube was placed on a magnetic rack for 2min to clarify the solution.
1.4.4.8. Mu.l of the supernatant was pipetted into a new PCR tube.
1.5 Bisulfite treatment and purification:
1.5.1. the desired reagent was taken out in advance and dissolved. The reagents were added according to the following table 10:
table 10
Component (A) High concentration sample (1 ng-2. Mu.g) volume Low concentration sample (1-500 ng) volume
Linker ligation of purified products 20μl 40μl
Bisulfite solution 85μl 85μl
DNA protection buffer 35μl 15μl
Total volume of 140μl 140μl
1.5.2_DNA protection buffer the addition of liquid turned blue. Gently blotted and mixed, and then split into two tubes for PCR.
1.5.3. The following procedure in table 11 was set and run: the lid was heated to 105 ℃.
TABLE 11
Temperature (temperature) Time of
95℃ min
60℃ 10min
95℃ min
60℃ 10min
4℃ ∞
1.5.4. The same sample from both tubes was combined into the same clean 1.5ml centrifuge tube by brief centrifugation.
1.5.5. To each sample, 310. Mu.l of buffer BL (sample size less than 100ng of 1. Mu.l of carrier RNA (1. Mu.g/. Mu.l) was added), vortexed, and briefly centrifuged.
1.5.6. 250 Μl of absolute ethanol was added to each sample, vortexed and mixed for 15s, centrifuged briefly, and the mixture was added to the prepared corresponding column.
1.5.7. Standing for 1min, centrifuging for 1min, transferring the liquid in the collecting pipe into a centrifugal column again, centrifuging for 1min, and discarding the liquid in the centrifugal pipe.
1.5.8. Add 500. Mu.l buffer BW (note whether absolute ethanol was added) centrifuge for 1min and discard the waste.
1.5.9. Add 500. Mu.l buffer BD (note whether absolute ethanol was added) cover the tube and leave it for 15min at room temperature. Centrifuging for 1min, and discarding the centrifuged liquid.
1.5.10. Mu.l of buffer BW (note whether absolute ethanol was added) was added, centrifuged for 1min, the detached liquid was discarded, and repeated 2 times.
1.5.11. 250 Μl of absolute ethanol was added, centrifuged for 1min, the column was placed in a new 2ml collection tube and all remaining liquid was discarded.
1.5.12. The column was placed in a clean 1.5ml centrifuge tube, 20. Mu.l of nuclease-free water was added to the center of the column membrane, the lid was gently covered, the column was placed at room temperature for 1min, and the column was centrifuged for 1min.
1.5.13. The liquid in the collection tube was re-transferred to a centrifuge column, left at room temperature for 1min, and centrifuged for 1min.
1.6. Pre-amplification and purification before hybridization:
1.6.1. the reaction system was prepared according to the following table 12, blown and mixed well, and centrifuged briefly:
Table 12
1.6.2. The procedure in table 13 below was set up and the PCR procedure was initiated: thermal cover 105 DEG C
TABLE 13
The number of PCR cycles was adjusted according to the amount of DNA to be added, and the reference data are shown in Table 14 below:
TABLE 14
1.6.4. 50 Mu l Agencourt AMPure XP magnetic beads are added into a PCR tube after the reaction is finished, and the mixture is blown and evenly mixed by a pipette to avoid generating bubbles (AgencourtAMPure XP is evenly mixed and balanced at room temperature in advance).
1.6.5. Incubating for 5-15min at room temperature, and placing the PCR tube on a magnetic rack for 3min to clarify the solution.
1.6.6. The supernatant was removed, the PCR tube was placed on a magnetic rack, 200. Mu.l of 80% ethanol solution was added to the PCR tube, and the mixture was allowed to stand for 30 seconds.
1.6.7. The supernatant was removed, 200. Mu.l of 80% ethanol solution was added to the PCR tube, and after standing for 30s, the supernatant was thoroughly removed (it was recommended to remove the bottom residual ethanol solution using a 10. Mu.l pipette).
1.6.8. Standing at room temperature for 5min to volatilize residual ethanol thoroughly.
1.6.9. Add 30. Mu.l of nuclease free water, remove the centrifuge tube from the magnetic rack and gently pipette the resuspended beads using a pipette.
1.6.10. Standing at room temperature for 2min, and placing 200 μl PCR tube on a magnetic rack for 2min to clarify the solution.
1.6.11. The supernatant was transferred to a new 200. Mu.l PCR tube (placed on an ice box) with a pipette, and the reaction tube was marked with a sample number, and prepared for the next reaction.
1.6.12. 1 Μl of the sample was used for library concentration determination using Qubit, and library concentration was recorded.
1.6.13. 1 Μl of the sample was used for library fragment length measurement using Agilent 2100, the library length being approximately between 270bp-320 bp.
1.7. Hybridization of sample to probe:
1.7.1. Sample libraries were mixed with various Hyb blockers, labeled B, according to the system in table 15 below:
TABLE 15
Component (A) Volume of
Pre-amplification product 750Ng of corresponding volume
Hyb human blockers 5μl
Joint blocking material 6μl
Reinforcing agent 5μl
1.7.2. The prepared mixture of the sample and the Hyb blocker is put into a vacuum concentration centrifuge, a PCR tube cover is opened, the centrifuge is started, a vacuum pump switch is opened, and concentration is started.
1.7.3. The drained sample was redissolved in about 9 μl of nuclease-free water, and mixed gently by pipetting, briefly centrifuged and placed on ice for use, labeled B.
1.7.4. And (3) placing the Hyb buffer solution in a room temperature for melting, wherein precipitation appears after melting, placing the mixture in a water bath at 65 ℃ for preheating after uniformly mixing, placing 20 mu l of the Hyb buffer solution (without precipitation and turbidity) in a new 200 mu l PCR tube after complete dissolution, covering a tube cover, marking as A, and continuously placing the tube cover in the water bath at 65 ℃ for incubation for later use.
1.7.5. The methylation probe sequence described previously was synthesized by Ai Jitai Kangshensu Biotech (Beijing) Inc.:
1.7.6. mu.l of the RNase-blocking material and 2. Mu.l of the probe composition were placed in a 200. Mu.l PCR tube, gently blotted and mixed, centrifuged briefly and placed on ice for use, labeled C.
1.7.7. Setting parameters of a PCR instrument, and heating the cover to 100 ℃,95 ℃ for 5min; and (5) maintaining at 65 ℃.
1.7.8. The PCR tube B was placed on a PCR instrument and the procedure was run.
When the temperature of the PCR instrument is reduced to 65 ℃, the PCR tube A is placed on the PCR instrument for incubation, and a thermal cover of the PCR instrument is covered.
After 1.7.10.5min, C was placed on PCR for incubation and covered with the thermal cover of the PCR instrument.
1.7.11. Placing the PCR tube C into a PCR instrument for 2min, adjusting the liquid transfer device to 13 μl, sucking 13 μl of Hyb buffer solution from the PCR tube A, transferring to the PCR tube C, sucking all samples in the PCR tube B, transferring to the PCR tube C, gently sucking for 10 times, mixing thoroughly, avoiding generating a large amount of bubbles, sealing the tube cover, covering the thermal cover of the PCR instrument, and incubating overnight at 65deg.C (16-24 h).
1.8. Capturing a target region DNA library:
1.8.1. Preparation of Capture magnetic beads
1.8.1.1. The beads (Dynabeads MyOne STREPTAVIDIN T magnetic beads) were removed from 4℃and resuspended by vortexing.
1.8.1.2. 50 Μl of magnetic beads were placed in a new PCR tube, placed on a magnetic rack for 1min to clarify the solution, and the supernatant was removed.
1.8.1.3. The PCR tube was removed from the magnetic rack, 200. Mu.L of binding buffer was added and gently pipetted several times to mix well and resuspend the beads.
1.8.1.4. Placing on a magnetic rack for 1min, and removing the supernatant.
1.8.1.5. Repeating the steps 3-4 twice, and washing the magnetic beads for 3 times.
1.8.1.6. The PCR tube was removed from the magnetic rack and 200. Mu.L of binding buffer was added to gently pipette 6 times to resuspend the beads for use.
1.8.2. Capturing a target DNA library
1.8.2.1. The hybridization product PCR tube C is kept on a PCR instrument, 200 mu L of prepared capture magnetic beads are added into the hybridization product PCR tube C, the hybridization product PCR tube C is sucked and beaten for 6 times by a pipette for uniform mixing, and the hybridization product PCR tube C is placed on a rotary mixer for 30min at room temperature (the rotating speed is preferably not more than 10 revolutions per minute).
1.8.2.2. The PCR tube was placed on a magnetic rack for 2min to clarify the solution and the supernatant was removed.
1.8.2.3. 200 Mu L of washing buffer 1 is added into the PCR tube C, gently sucked and beaten for 6 times of uniform mixing, placed on a rotary mixer for cleaning for 15min (the rotating speed is preferably not more than 10 revolutions per minute), then centrifuged briefly, the PCR tube is placed on a magnetic rack for 2min to clarify the solution, and the supernatant is removed.
1.8.2.4. 200 Μl of washing buffer 2 preheated at 65deg.C is added, gently sucked and beaten for 6 times, mixed well, placed on a mixing instrument, incubated at 65deg.C for 10min, and washed at 800 rpm.
1.8.2.5. The PCR tube was placed on a magnetic rack for 2min after brief centrifugation and the supernatant removed. The washing with wash buffer 2 was repeated 2 more times for a total of 3 times. The wash buffer 2 was removed thoroughly last time.
The PCR tube was placed on a magnetic rack, 200. Mu.l of 80% ethanol was added to the PCR tube, and after standing for 30 seconds, the ethanol solution was thoroughly removed and dried at room temperature for 2 minutes.
1.8.2.7. Adding 30 mu L nuclease-free water into the PCR tube, taking the PCR tube off the magnetic rack, and lightly sucking and beating the magnetic beads for 6 times for later use.
1.9. Post-capture amplification and purification
1.9.1. The reaction system was prepared according to the following table 16 to enrich the captured library, and after gently stirring and mixing, the reaction system was centrifuged briefly:
Table 16
1.9.2. The following procedure in table 17 was set, samples were placed in a PCR instrument, and the procedure was run: the lid was heated to 105 ℃.
TABLE 17
After the PCR was completed, 55. Mu. l Agencourt AMPure XP magnetic beads were added to the sample, and the mixture was gently pipetted and mixed.
1.9.4. Incubation was performed for 5min at room temperature, and the PCR tube was placed on a magnetic rack for 3min to clarify the solution.
1.9.5. The supernatant was removed, the PCR tube was placed on a magnetic rack, 200. Mu.l of 80% absolute ethanol was added, and the mixture was allowed to stand for 30 seconds.
1.9.6. The supernatant was removed, 200. Mu.l of 80% absolute ethanol was added to the PCR tube, and the supernatant was thoroughly removed after standing for 30.
1.9.7. Standing at room temperature for 5min to volatilize residual ethanol thoroughly.
1.9.8. Add 25. Mu.l of nuclease-free water, remove the PCR tube from the magnetic rack, gently blow mix and re-suspend the beads and leave for 2min at room temperature.
1.9.9. The PCR tube was placed on a magnetic rack for 2min to clarify the solution.
1.9.10. Mu.l of the supernatant was pipetted into a 1.5ml centrifuge tube and labeled with sample information.
1.9.11. 1 Μl of library was quantified using Qubit and library concentrations were recorded.
1.9.12. 1 Μl of sample was taken and used for library fragment length determination using Agilent 2100.
1.9.13. Sequencing was performed using Illumina high throughput sequencing platform.
1.10. Methylation letter analysis flow. The method is approximately as follows: checking sequencing quality by using fastp quality control software, removing low-quality reads, comparing the quality-controlled clean data to a reference genome by using Bismark comparison software, and extracting corresponding methylation sites by using Bismar _methyl_ extractor software. Finally, the methylation level of the target region is calculated, and the value results in a diagnosis of cancer if the threshold value is exceeded and a diagnosis of normal if the threshold value is lowered.
Example 2
Based on 40 samples clinically diagnosed as liver cancer collected from Beijing area and 40 healthy human samples collected from Beijing area, the methylation levels of the 12 methylation biomarkers screened were calculated using the methylation library building method described in example 1, and the threshold (hereinafter referred to as site or marker) and the separately discriminated AUC values were calculated from the methylation levels of the 12 methylation biomarkers in the liver cancer blood sample and normal human blood sample data sets as shown in Table 18. In addition, we performed a joint interpretation of the 3 markers with the greatest AUC [ methylation level ], using a generalized linear regression model. The generalized linear regression construction model is: logp/(1-p) =1.317× SEQ ID NO.3+1.029*SEQ ID NO.4+0.052*SEQ ID NO.5, where p is the probability that the predicted sample is lung cancer. When the model calculates the subject prediction score [ i.e.: logp/(1-p) exceeding 0.4 is judged to be cancerous. The sensitivity of model predictions at 10X cross validation was 100% [78% -100% ], the specificity was 100% [78% -100% ], and AUC was 0.99, as shown in table 19.
Table 1812 data on the specific expression of the single methylation markers
SEQ ID Threshold value Specificity Sensitivity AUC
SEQ ID NO.1 0.32 0.8 0.93 0.89
SEQ ID NO.2 0.12 0.73 1 0.88
SEQ ID NO.3 0.12 0.93 0.86 0.96
SEQ ID NO.4 0.21 0.93 0.93 0.98
SEQ ID NO.5 0.04 0.86 0.93 0.96
SEQ ID NO.6 0.05 0.93 0.73 0.9
SEQ ID NO.7 0.19 0.93 0.8 0.91
SEQ ID NO.8 0.09 0.86 0.86 0.89
SEQ ID NO.9 0.1 0.88 0.85 0.9
SEQ ID NO.10 0.15 0.85 0.95 0.92
SEQ ID NO.11 0.2 0.9 0.9 0.94
SEQ ID NO.12 0.14 0.93 0.9 0.95
Table 193 data on the specific performance of methylation marker combinations
Example 3
20 Human samples (S1-10 is a healthy human sample, S11-20 is a liver cancer patient sample), and peripheral blood is collected by adopting the methylation marker detection method according to the application according to the method of the embodiment 1; establishing a library, and sequencing through an Illumina platform; sequencing data is subjected to the biological information analysis flow to obtain the methylation level of each marker, the disease condition of the patient is predicted according to the threshold value of each marker, if the patient is a cancer sample if the patient exceeds the threshold value, and if the patient is a healthy human sample if the patient falls below the threshold value, the specific results are shown in the following table 20,SEQ ID NO.3&SEQ ID NO.4&SEQ ID NO.5, and the predicted results of the 3 combined methylation markers in the 20 samples are shown in the table 21:
Wherein, the interpretation result, 0, represents the classification as normal, i.e. healthy; 1 represents a classification as abnormal, i.e. tumor.
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Table 213 prediction results for the Joint methylation markers in 20 samples
SEQ ID SEQ ID NO.3&SEQ ID NO.4&SEQ ID NO.5
S1 predictive p-value 0.12
S1 interpretation result 0
S2 predictive p-value 0.17
S2 interpretation result 0
S3 predictive p-value 0.19
S3 interpretation result 0
S4 predictive p-value 0.22
S4 interpretation result 0
S5 predictive p-value 0.3
S5 interpretation result 0
S6 predictive p-value 0.24
S6 interpretation result 0
S7 predictive p-value 0.28
S7 interpretation result 0
S8 predictive p-value 0.19
S8 interpretation result 0
S9 predictive p-value 0.25
S9 interpretation result 0
S10 predictive p-value 0.38
S10 interpretation result 0
S11 predictive p-value 0.44
S11 interpretation result 1
S12 predictive p-value 0.52
S12 interpretation result 1
S13 predictive p-value 0.66
S13 interpretation result 1
S14 predictive p-value 0.51
S14 interpretation result l
S15 predictive p-value 0.49
S15 interpretation result 1
S16 predictive p-value 0.48
S16 interpretation result 1
S17 predictive p-value 0.87
S17 interpretation results 1
S18 predictive p-value 0.47
S18 interpretation result 1
S19 predictive p-value 0.59
S19 interpretation results 1
S20 predictive p-value 0.65
S20 interpretation result 1
In summary, the inventors of the present application have obtained methylation genes associated with liver cancer, determined marker sequences of methylation abnormality of liver cancer methylation genes, and, through the marker sequences of these methylation genes, can sensitively and specifically detect the methylation state of the genes, thereby being useful for detecting peripheral blood episomal DNA.
Although the embodiments of the present application have been described above in connection with the above, the present application is not limited to the above-described specific embodiments and fields of application, which are merely illustrative, instructive, and not restrictive. Those skilled in the art, having the benefit of this disclosure, may effect numerous forms of the application without departing from the scope of the application as claimed.
The main sequence table is shown in table 22:
Table 22
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Claims (10)

1. A marker for detecting liver cancer, the marker being selected from one or more of the following genes: PSD4, MIR4783, PLAC8, TSTYL 5, FAR1, PPFIA1, KIF7.
2. The marker according to claim 1, wherein the nucleotide sequence of the marker is selected from one of the markers shown in SEQ ID No. 1-SEQ ID No. 12, preferably the marker is a methylated marker.
3. A probe composition for detecting liver cancer, the probe composition comprising a nucleic acid that targets methylation of the marker of claim 1 or 2.
4. A probe composition according to claim 3, wherein the probe composition comprises a hypermethylated first probe composition for hybridization with a bisulfite converted CG hypermethylated region and a hypomethylated second probe composition for hybridization with a bisulfite converted CG hypomethylated region;
preferably, the first probe composition comprises n probes that hybridize to each nucleotide of the sense strand and/or the antisense strand of the bisulfite-converted CG hypermethylated region;
preferably, the second probe composition comprises m probes that hybridize to each nucleotide of the sense strand and/or the antisense strand of the bisulfite-converted CG hypomethylated region;
preferably, n and m are each any integer from 1 to 10;
Preferably, there is an overlap of x 1 nucleotides between the n-1 th probe and the n-th probe, preferably x 1 is any integer from 0 to 100;
Preferably, there is an overlap of x 2 nucleotides between the m-1 th probe and the m-th probe, preferably x 2 is any integer from 0 to 100;
Further preferably, the first probe composition comprises two or four or six or eight or ten or twelve of SEQ ID NOS: 61-88 and the second probe composition comprises two or four or six or eight or ten or twelve of SEQ ID NOS: 89-116.
5. Use of a nucleic acid molecule for detecting a marker selected from one or more of the following genes in the preparation of a kit for detecting liver cancer: PSD4, MIR4783, PLAC8, TSTYL 5, FAR1, PPFIA1, KIF7.
6. Use according to claim 5, wherein the nucleotide sequence of the target sequence of the marker is selected from one of the markers shown in SEQ ID NOs 1-9, preferably the marker is a methylated marker;
preferably, the probe composition is used for targeting a methylated marker of liver cancer;
preferably, the probe composition is the probe composition of claim 3 or 4.
7. A composition for liver cancer detection, the composition comprising a nucleic acid for detecting a methylation state of any one or more markers selected from the group consisting of: PSD4, MIR4783, PLAC8, TSTYL 5, FAR1, PPFIA1, KIF7, preferably the nucleotide sequence of the marker is selected from one of the nucleotide sequences shown in SEQ ID NO 1-SEQ ID NO 12.
8. The composition of claim 7, wherein the nucleic acid comprises the probe composition of claim 3 or 4;
Preferably, the nucleic acid comprises:
A primer that is a fragment of at least 9 nucleotides in the marker, the fragment comprising at least one CpG dinucleotide sequence;
Preferably, the nucleic acid further comprises:
A probe that hybridizes under moderately stringent or stringent conditions to at least 15 nucleotide fragments in said marker, said fragments comprising at least one CpG dinucleotide sequence;
Preferably, the composition further comprises an agent that converts the unmethylated cytosine base at position 5 of the marker to uracil;
Preferably, the nucleic acid for detecting methylation of a marker further comprises:
Blocking agents that preferentially bind to target sequences in the unmethylated state.
9. A kit comprising reagents for detecting the marker of claim 1 or 2 or the probe composition of claim 3 or 4 or the composition of claim 7 or 8.
10. A chip comprising a reagent for detecting the marker of claim 1 or 2 or the probe composition of claim 3 or 4 or the composition of claim 7 or 8.
CN202211442630.5A 2022-11-17 2022-11-17 Marker for screening liver cancer, probe composition and application thereof Pending CN118048451A (en)

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