CN114395626B - Marker for cervical cancer screening, probe composition and application thereof - Google Patents

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

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CN114395626B
CN114395626B CN202210005178.XA CN202210005178A CN114395626B CN 114395626 B CN114395626 B CN 114395626B CN 202210005178 A CN202210005178 A CN 202210005178A CN 114395626 B CN114395626 B CN 114395626B
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CN114395626A (en
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关晋霞
韩晓亮
连明
张聪
王小奇
吴宁宁
李永君
叶志海
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Biochain Beijing Science and Technology Inc
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Abstract

The invention discloses a marker for cervical cancer screening, a probe composition and application thereof, wherein the marker is selected from any one of 9 markers. 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 cervical cancer screening, probe composition and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a marker for cervical cancer screening, a probe composition and application thereof.
Background
Cervical cancer is one of the most common types of gynecological cancers, according to the 2020 cancer statistics, about 604,127 new cases of cervical cancer and about 341,000 death cases are seen in the global cases, and the 4 th new cases and the 4 th death rate of women are all ranked. Over 80% of cervical cancer cases worldwide occur in developing and underdeveloped countries. The new cervical cancer cases in 2020 are about 11 ten thousand and the death cases are about 6 ten thousand. Epidemiological data indicate that persistent infection with high risk human papillomaviruses (human papillomavirus, HPV) is a major risk factor for cervical cancer development. When the body is infected with HPV an immune response is generated, during which most HPV infections do not cause clinical manifestations in its host, and most are cleared after a short period of time, but still-10% persist. Changes from HPV-infected cervical intraepithelial neoplasia to carcinogenesis are a slow process, for years or even decades. In 2018, WHO initiated a global strategy to eliminate cervical cancer, aimed at reducing the global cervical cancer incidence to 4/10 ten thousand. Crowd screening is one of the major interventions facilitating the implementation of this project.
Clinical staging in cancer diagnosis is a major determinant of survival of cancer patients. And the cancer screening can realize the prevention and early diagnosis and early intervention of the cancer, thereby improving the survival rate of 5 years. The current clinical cervical cancer screening method comprises: cytological screening, colposcopy, cervical imaging and HPV DNA detection. Although the use of these screening methods has greatly reduced the incidence and mortality of cervical cancer, a significant number of cervical cancer patients remain missed or overstocked due to the complex environment within the uterus and the quality of the detection methods. For example, cytological screening results rely on cervical cell smear quality and cytological expert experience, resulting in a less sensitive method. The HPV DNA technology can effectively improve the sensibility of HPV infection induced cervical cancer and reduce colposcopy. However, since the method cannot accurately identify pathogenic HPV infection and transient infection, and thus high false positive results are produced, the transfer colposcopy is over-medical. In addition, as the screening methods are invasive, have high cost and influence compliance, and are not beneficial to the screening of cervical cancer patients. Therefore, there is a need in the clinic for an early cervical cancer prevention and diagnosis tool that combines high sensitivity and specificity, with high objective accuracy, and facilitates the popularization from opportunistic screening to population screening.
Epigenetic studies have shown that specific DNA methylation abnormalities promote cancer occurrence by inhibiting transcriptional inactivation of oncogenes. For example, CDH1 has now been found to exhibit hypermethylation in most primary cervical cancers. However, there is currently no methylation biomarker for clinical screening or diagnosis of cervical cancer. DNA methylation has been demonstrated to be tissue specific, utilizing free DNA methylation signals in the blood for early cancer detection, and can be traced to tumor origins based on circulating tumor DNA (ctDNA) methylation signatures. It is expected that it will significantly improve health results, reduce the occurrence of diseases such as cervical cancer that can be found early, and eventually may improve quality of life health.
Disclosure of Invention
The invention aims to provide a marker for detecting cervical cancer and a probe composition, which can be used for screening cervical cancer, wherein the marker is used for screening asymptomatic people in a non-invasive mode and prognosis detection of cancer patients, so that the damage caused by invasive detection is reduced, and the sensitivity and the accuracy are higher.
The specific technical scheme of the invention is as follows:
1. a marker for detecting cervical cancer, wherein the marker is selected from one of the following: LINC00643, ZNF773, CITED1, GABRA1, BCOR, NXPH1, DIAPH2, FAM133A and BCOR.
2. The marker according to item 1, wherein the nucleotide sequence of the marker is selected from one of the markers shown in SEQ ID NO. 1-9, preferably the marker is a methylated marker.
3. A probe composition comprising a probe that targets methylation of a 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 to a bisulfite converted CG hypermethylated region and a hypomethylated second probe composition for hybridization to a bisulfite converted CG hypomethylated region;
preferably, the first probe composition comprises n probes that hybridize to each nucleotide of the sense and antisense strands of the bisulfite converted CG hypermethylated region;
preferably, the second probe composition comprises m probes that hybridize to each nucleotide of the sense and antisense strands of the bisulfite converted CG hypomethylated region;
preferably, n and m are each any integer from 1 to 10;
Preferably, there is x between the n-1 th probe and the n-th probe 1 Overlapping of nucleotides, preferably x 1 Is any integer from 0 to 100;
preferably, there is x between the m-1 th probe and the m-th probe 2 Overlapping of nucleotides, preferably x 2 Is any integer from 0 to 100;
further preferably, the first probe composition comprises one or two nucleotide sequences as shown in SEQ ID NOS.10-27 and the second probe composition comprises one or two nucleotide sequences as shown in SEQ ID NOS.28-45.
5. Use of a marker for the manufacture of a kit for the detection of cervical cancer, characterized in that the marker is selected from one of the following: LINC00643, ZNF773, CITED1, GABRA1, BCOR, NXPH1, DIAPH2, FAM133A and BCOR.
6. The use according to item 5, characterized in that the nucleotide sequence of the marker is selected from one of the markers shown in SEQ ID NO. 1-9, preferably the marker is a methylated marker;
preferably, the probe composition is used for targeting a methylated marker of cervical cancer;
preferably, the probe composition is the probe composition according to item 3 or 4.
7. A composition for cervical cancer detection, comprising a nucleic acid for detecting methylation of any one of the markers selected from the group consisting of: LINC00643, ZNF773, CITED1, GABRA1, BCOR, NXPH1, DIAPH2, FAM133A and BCOR.
8. The composition of item 7, wherein the nucleotide sequence of the marker is selected from one of the nucleotide sequences set forth in SEQ ID NOs 1-9.
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 a target sequence of 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 a target sequence of the marker, the 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 target sequence of the marker to uracil;
preferably, the nucleic acid for detecting methylation of a target sequence of a marker further comprises:
blocking agents that preferentially bind to target sequences in the unmethylated state.
10. A kit comprising the marker of item 1 or 2 or the probe composition of item 3 or 4 or the composition of any one of items 7-9.
11. A chip comprising the marker of item 1 or 2 or the probe composition of item 3 or 4 or the composition of item 7 or 8.
ADVANTAGEOUS EFFECTS OF INVENTION
The invention utilizes the epigenomic and bioinformatics technology, finds a plurality of methylation genes related to cervical cancer by analyzing genome methylation data of the cervical cancer, determines a target sequence of methylation abnormality of the methylation genes of the cervical 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
The present invention will be described in detail below. While specific embodiments of the invention are shown, it should be understood that the invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
It should be noted that certain terms are used throughout the description and claims to refer to particular components. Those of skill in the art will understand that a person may refer to the same component by different names. The specification and claims do not identify differences in terms of components, but rather differences in terms of the functionality of the components. As referred to throughout the specification and claims, the terms "include" or "comprising" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description hereinafter sets forth a preferred embodiment for practicing the invention, but is not intended to limit the scope of the invention, as the description proceeds with reference to the general principles of the description. The scope of the invention is defined by the appended claims.
The invention provides a marker for detecting cervical cancer, which is selected from one of the following: LINC00643, ZNF773, CITED1, GABRA1, BCOR, NXPH1, DIAPH2, FAM133A and BCOR.
In a specific embodiment, the nucleotide 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.
Wherein, the nucleotide sequence of LINC00643 is shown as SEQ ID NO. 1; the nucleotide sequence of ZNF773 is shown as SEQ ID NO. 2; the nucleotide sequence of CITED1 is shown as SEQ ID NO. 3; the nucleotide sequence of GABRA1 is shown as SEQ ID NO. 4; the nucleotide sequence of BCOR is shown as SEQ ID NO. 5; the nucleotide sequences of the NXPH1 are respectively shown in SEQ ID NO. 6; the nucleotide sequences of the DIAPH2 are respectively shown in SEQ ID NO. 7; the nucleotide sequence of FAM133A is shown in SEQ ID NO. 8; the nucleotide sequence of BCOR is shown as SEQ ID NO. 9.
Wherein the sequences of the markers are all sequences which are not converted by bisulfite.
The invention provides a probe composition comprising a probe that targets methylation of the marker.
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 replication process of DNA under the action of DNA methyltransferase, so that the methylation of the gene promoter region can lead to silence transcription of cancer suppressor genes during DNA methylation, and the methylation is closely related to 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, and can be combined with (hybridized with) complementary unlabeled single-stranded DNA or RNA in a sample to be tested in a hydrogen bond manner to form a double-stranded complex (hybrid). 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 a specific embodiment, the probe composition comprises a hypermethylated first probe composition for hybridization to a bisulfite converted CG hypermethylated region and a hypomethylated second probe composition for hybridization to a bisulfite converted CG hypomethylated region.
The hypermethylation means that after the marker is converted by bisulfite, a base C is changed into a base T, but if the marker is a base CG, the base C is kept unchanged;
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 T.
Since the methylation status varies from person to person, the sequence of the tag converted by bisulfite varies, one extreme case of each tag is shown here, i.e. all CG of the segment is in hypermethylation status, and the hypermethylation status sequence of its complementary strand:
the sequence of one extreme case of SEQ ID NO. 1 is shown as SEQ ID NO. 46;
the complement of the sequence in the extreme case is shown as SEQ ID NO. 47;
the sequence of one extreme case of SEQ ID NO. 2 is shown as SEQ ID NO. 48;
the complement of the sequence in the extreme case is shown as SEQ ID NO. 49;
the sequence of one extreme case of SEQ ID NO. 3 is shown as SEQ ID NO. 50;
the complementary strand of the sequence in the extreme case is shown as SEQ ID NO. 51;
the sequence of one extreme case of SEQ ID NO. 4 is shown as SEQ ID NO. 52;
the complementary strand of the sequence in the extreme case is shown in SEQ ID NO. 53;
The sequence of one extreme case of SEQ ID NO. 5 is shown as SEQ ID NO. 54;
the complement of the sequence in the extreme case is shown as SEQ ID NO. 55;
the sequence of one extreme case of SEQ ID NO. 6 is shown as SEQ ID NO. 56;
the complement of the sequence in the extreme case is shown as SEQ ID NO. 57;
the sequence of one extreme case of SEQ ID NO. 7 is shown as SEQ ID NO. 58;
the complement of the sequence in the extreme case is shown as SEQ ID NO. 59;
the sequence of one extreme case of SEQ ID NO. 8 is shown as SEQ ID NO. 60;
the complementary strand of the sequence in the extreme case is shown in SEQ ID NO. 61;
the sequence of one extreme case of SEQ ID NO. 9 is shown as SEQ ID NO. 62;
the complement of the sequence in the extreme case is shown in SEQ ID NO. 63.
Similarly, since each person has a different methylation state, an extreme case is shown here, in which all CG is in hypomethylated state, and the sequence of hypomethylated states of their complementary strands is also shown:
the sequence of one extreme case of SEQ ID NO. 1 is shown as SEQ ID NO. 64;
the complementary strand of the sequence in the extreme case is shown as SEQ ID NO. 65;
the sequence of one extreme case of SEQ ID NO. 2 is shown as SEQ ID NO. 66;
the complement of the sequence in the extreme case is shown as SEQ ID NO. 67;
The sequence of one extreme case of SEQ ID NO. 3 is shown as SEQ ID NO. 68;
the complement of the sequence in the extreme case is shown as SEQ ID NO. 69;
the sequence of one extreme case of SEQ ID NO. 4 is shown as SEQ ID NO. 70;
the complementary strand of the sequence in the extreme case is shown as SEQ ID NO. 71;
the sequence of one extreme case of SEQ ID NO. 5 is shown as SEQ ID NO. 72;
the complement of the sequence in the extreme case is shown as SEQ ID NO. 73;
the sequence of one extreme case of SEQ ID NO. 6 is shown as SEQ ID NO. 74;
the complement of the sequence in the extreme case is shown as SEQ ID NO. 75;
the sequence of one extreme case of SEQ ID NO. 7 is shown as SEQ ID NO. 76;
the complement of the sequence in the extreme case is shown as SEQ ID NO. 77;
the sequence of one extreme case of SEQ ID NO. 8 is shown as SEQ ID NO. 78;
the complement of the sequence in the extreme case is shown as SEQ ID NO. 79;
the sequence of one extreme case of SEQ ID NO. 9 is shown as SEQ ID NO. 80;
the complement of the sequence in the extreme case is shown in SEQ ID NO. 81.
In a specific embodiment, the first probe composition comprises n probes that hybridize to each nucleotide of the sense and antisense strands of the bisulfite converted CG hypermethylated region.
The second probe composition includes m probes that hybridize to each nucleotide of the sense and antisense strands of the bisulfite converted CG hypomethylated region.
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 a specific embodiment, there is x between the n-1 th probe and the n-th probe 1 Overlapping of nucleotides, preferably x 1 Is any integer from 0 to 100;
preferably, there is x between the m-1 th probe and the m-th probe 2 Overlapping of nucleotides, preferably x 2 Is any integer from 0 to 100.
Wherein x is 1 And x 2 May be the same or different, when x 1 When 0, it is indicated that the tail of the n-1 th probe is connected with the head of the n-th probe, and similarly, when x 2 When 0, it is indicated that the tail of the m-1 th probe is connected to the head of the m-th probe.
According to the invention, the probe composition is hybridized with the marker converted by the bisulfite, wherein the hypermethylated first probe composition is hybridized with the CG hypermethylated region, and the hypomethylated second probe composition is hybridized with the CG hypomethylated region, so that the methylation level of the target sequence can be detected efficiently and accurately, and the probe composition can be used for cervical cancer screening.
In a specific embodiment, the hypermethylated first probe composition comprises one or both of SEQ ID NOS: 10-27.
The hypomethylated second probe composition comprises one or two of SEQ ID NOS.28-45.
Wherein the first probe composition for hybridization with LINC00643 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NO. 10-11;
the first probe composition for hybridization with ZNF773 methylation sequence comprises the nucleotide sequence shown in SEQ ID NOS.12-13;
the first probe composition for hybridization with the CITED1 methylation sequence comprises a nucleotide sequence as shown in SEQ ID NOS.14-15;
the first probe composition for hybridization to a GABRA1 methylated sequence comprises the nucleotide sequence set forth in SEQ ID NOS.16-17;
the first probe composition for hybridization with BCOR methylation sequences comprises the nucleotide sequence shown as SEQ ID NOS.18-19;
the first probe composition for hybridization with a NXPH1 methylation sequence comprises the nucleotide sequence shown in SEQ ID NOS.20-21;
the first probe composition for hybridization to a DIAPH2 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS.22-23;
a first probe composition for hybridization to FAM133A methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS.24-25;
The first probe composition for hybridization with BCOR methylation sequences comprises the nucleotide sequences shown as SEQ ID NOS.26-27;
a second probe composition for hybridization to LINC00643 methylation sequences comprises the nucleotide sequence shown in SEQ ID NOS.28-29;
a second probe composition for hybridization with a ZNF773 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS.30-31;
a second probe composition for hybridization with a CITED1 methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS.32-33;
a second probe composition for hybridization to a GABRA1 methylated sequence comprises the nucleotide sequence set forth in SEQ ID NOS.34-35;
the second probe composition for hybridization with BCOR methylation sequences comprises the nucleotide sequence shown as SEQ ID NOS.36-37;
a second probe composition for hybridization to a NXPH1 methylation sequence comprises the nucleotide sequences shown in SEQ ID NOS.38-39;
a second probe composition for hybridization to DIAPH2 methylation sequences comprises the nucleotide sequences shown in SEQ ID NOS.40-41;
a second probe composition for hybridization to FAM133A methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS.42-43;
a second probe composition for hybridization with a BCOR methylation sequence comprises the nucleotide sequence set forth in SEQ ID NOS.44-45.
The invention provides application of a marker in preparation of a kit for detecting cervical cancer, wherein the marker is selected from one of the following: LINC00643, ZNF773, CITED1, GABRA1, BCOR, NXPH1, DIAPH2, FAM133A and BCOR.
In a specific embodiment, the nucleotide 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.
The invention provides application of a probe composition in preparation of a kit for detecting cervical cancer, wherein the probe composition is used for targeting a marker after methylation of the cervical cancer.
In a specific embodiment, the probe composition is the probe composition described above.
The present invention provides a composition for cervical cancer detection comprising a nucleic acid for detecting methylation of any one of the markers selected from the group consisting of: LINC00643, ZNF773, CITED1, GABRA1, BCOR, NXPH1, DIAPH2, FAM133A and BCOR, preferably, the nucleotide sequence of the marker is selected from one of the nucleotide sequences shown in SEQ ID NO 1-9.
In a specific embodiment, the nucleic acid comprises a probe composition as described above.
In a specific embodiment, the nucleic acid comprises:
A primer that is a fragment of at least 9 nucleotides in a target sequence of the marker, the fragment comprising at least one CpG dinucleotide sequence.
Wherein, if bisulfite is used to convert NDA in a sample to be tested, the nucleic acid for detecting methylation of the target sequence 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 a specific embodiment, the nucleic acid further comprises:
a probe that hybridizes under moderately stringent or stringent conditions to at least 15 nucleotide fragments in a target sequence of the marker, the fragments comprising at least one CpG dinucleotide sequence.
In a specific embodiment, the composition further comprises an agent that converts the unmethylated cytosine base at position 5 of the target sequence of the marker to uracil, e.g., the agent can be bisulfite or the like; preferably, the nucleic acid for detecting methylation of a target sequence 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 preferentially binds to the blocker, and thus the DNA template binds to the PCR obligation, and thus PCR amplification does not occur, whereas the DNA in the methylated state does not bind to the blocker and thus the primer set, PCR amplification occurs, and then the fragment obtained by the amplification is detected directly or indirectly.
The invention provides a kit comprising the above marker or the above probe composition or the above composition.
In a specific embodiment, the kit further comprises a container for holding a biological sample of the subject.
In a specific 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 the 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 a methylation level of a target sequence of a marker using the above-described kit, comprising the steps of:
(1) Extracting peripheral blood of a subject, and separating plasma or serum;
(2) Extracting free DNA from plasma or serum;
(3) Treating the free DNA obtained in step (2) with a reagent to convert the unmethylated cytosine base at position 5 to uracil or other bases, i.e., to convert the unmethylated cytosine base at position 5 of the target sequence 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 target sequence of the marker such that the target sequence of the treated marker is amplified to produce amplified products or not amplified; the target sequence of the treated marker, if subjected to DNA polymerization, produces amplification products; the target sequence of 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 target sequence of the marker based on the presence or absence of the amplification product, thereby determining the methylation level of the target sequence of the marker.
The invention provides a chip comprising the marker or the probe composition or the composition.
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 invention provides a cervical cancer screening method, which comprises the following steps:
detecting the methylation level of the marker, and
determining the risk of the subject for cervical cancer based on the methylation level, the marker being selected from one of:
LINC00643, ZNF773, CITED1, GABRA1, BCOR, NXPH1, DIAPH2, FAM133A and BCOR.
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 it involved 7769 cancer tissue samples of 26 tumors altogether, 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), cervical carcinoma (286), 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 (Whole Genome Bisulfite Sequencing, WGBS).
2) Candidate marker screening: for healthy plasma samples, the third quartile (Q3), also known as the "greater quartile", of the beta value of each probe corresponding to the 450K corresponding region was calculated, and the sites with Q3<0.02 were screened, resulting in List1. For 450K chip organization data, calculating a first quartile (Q1) of beta value of each probe corresponding to the 450K corresponding region, wherein Q1 is also called as smaller quartile, screening sites of Q1>0.1, and obtaining a result as List2. Taking the intersection of List1 and List2 yields 65739 differentially methylated regions.
3) And (3) marker selection: among the above markers, markers specific to cervical cancer were selected to obtain 43 markers. Meanwhile, the difference between methylation levels of 450k chip cervical cancer tissue (286) and beside cancer tissue (5) in TCGA is more than 0.2, and finally 35 different methylation areas are obtained.
4) And (3) marker verification: the probes are designed for capturing the 35 different methylation areas, and the data of the Bohr hong plasma samples (the number of cervical cancer samples=20 and the number of healthy people samples=24) are utilized for verification, so that 9 markers capable of distinguishing cervical cancer from healthy people are finally obtained. The sequences are shown in SEQ ID NO 1-9.
Customizing a probe composition (panel) comprising a hypermethylated first probe composition and a hypomethylated second probe composition according to the obtained target sequence region, wherein the first probe composition comprises two probes for each marker, and the first probe composition comprises a nucleotide sequence as shown in SEQ ID NO. 10-11 for SEQ ID NO. 1;
For SEQ ID NO. 2, the first probe composition comprises the nucleotide sequence shown as SEQ ID NO. 12-13; for SEQ ID NO. 3, the first probe composition comprises the nucleotide sequence shown as SEQ ID NO. 14-15; for SEQ ID NO. 4, the first probe composition comprises the nucleotide sequence shown as SEQ ID NO. 16-17; for SEQ ID NO. 5, the first probe composition comprises the nucleotide sequence shown as SEQ ID NO. 18-19; for SEQ ID NO. 6, the first probe composition comprises the nucleotide sequence shown as SEQ ID NO. 20-21; for SEQ ID NO. 7, the first probe composition comprises the nucleotide sequence shown as SEQ ID NO. 22-23; for SEQ ID NO. 8, the first probe composition comprises the nucleotide sequence shown as SEQ ID NO. 24-25; for SEQ ID NO. 9, the first probe composition comprises the nucleotide sequences as shown in SEQ ID NO. 26-27.
The second probe composition comprises two probes, wherein for SEQ ID NO. 1, the second probe composition comprises the nucleotide sequences as shown in SEQ ID NO. 28-29; for SEQ ID NO. 2, the second probe composition comprises the nucleotide sequence shown as SEQ ID NO. 30-31; for SEQ ID NO. 3, the second probe composition comprises the nucleotide sequence shown as SEQ ID NO. 32-33; for SEQ ID NO. 4, the second probe composition comprises the nucleotide sequence shown as SEQ ID NO. 34-35; for SEQ ID NO. 5, the second probe composition comprises the nucleotide sequence shown as SEQ ID NO. 36-37; for SEQ ID NO. 6, the second probe composition comprises the nucleotide sequence shown as SEQ ID NO. 38-39; for SEQ ID NO. 7, the second probe composition comprises the nucleotide sequence shown as SEQ ID NO. 40-41; for SEQ ID NO. 8, the second probe composition comprises the nucleotide sequence shown as SEQ ID NO. 42-43; for SEQ ID NO. 9, the second probe composition comprises the nucleotide sequences as shown in SEQ ID NO. 44-45.
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 the other one used in the experiment.
TABLE 1
1.1.2. Cleavage and binding
1.1.2.1. The binding solution/bead mixture was prepared according to the following table 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. The centrifuge tube containing the bead resuspension was placed on a magnetic rack for 20s.
1.1.3.4. The separated supernatant was aspirated and the binding tube was washed, and the washed residual beads were collected again into a heavy suspension, discarding the lysis/binding tube.
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 according to the following table.
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 of gDNA was taken, added with water to 125. Mu.l, added to a covaries 130. Mu.l disruption 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 the mixture was supplemented to 50. Mu.l with nuclease-free water, and the following reagents 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 was set up for the reaction on the PCR instrument: the temperature of the hot cover is 85 ℃.
TABLE 5
Temperature (temperature) Time
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 the following table:
TABLE 6
1.4.2. The following reagents were prepared according to the following table, gently blotted and mixed, and centrifuged briefly:
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 was set up for the reaction on the PCR instrument: there is no thermal cover.
TABLE 8
Temperature (temperature) Time
20℃ 30min
4℃ ∞
1.4.4. Adding purified magnetic beads for experiment (Agencourt AMPure XP magnetic beads are taken to room temperature in advance, and are vibrated and mixed uniformly for standby) according to the following system:
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. 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.
1.4.4.4. 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:
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
The DNA protection buffer was added to the liquid to turn blue. Gently blotted and mixed, and then split into two tubes for PCR.
1.5.3. The following procedure was set up and run: the lid was heated to 105 ℃.
TABLE 11
Temperature (temperature) Time
95℃ 5min
60℃ 10min
95℃ 5min
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. preparing a reaction system according to the following table, blowing, mixing uniformly and centrifuging briefly:
table 12
1.6.2. The following procedure was set and the PCR procedure was started: 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 were as follows:
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 (Agencourt AMPure 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 following system:
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 before was synthesized by Ai Jitai c biotechnology (beijing) limited:
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 T1) were removed from 4 ℃, 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 (23.5 ml of nuclease-free water, 1.25ml of 20 XSSC, 250. Mu.L of 10% SDS) was added to the PCR tube C, gently blotted and homogenized, placed on a rotary kneader and washed for 15min (the rotation speed is preferably not more than 10 rpm), and then centrifuged briefly, and the PCR tube was placed on a magnetic rack for 2min to clarify the solution, and the supernatant was removed.
1.8.2.4. 200. Mu.l of washing buffer 2 (24.6 ml of nuclease-free water, 125. Mu.l of 20 XSSC, 250. Mu.l of 10% SDS) preheated at 65℃was added, gently blotted 6 times and mixed, and incubated on a mixer at 65℃for 10min at a rotational speed of 800 rpm for washing.
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. Preparing a reaction system according to the following table, enriching a capture library, lightly blowing and uniformly mixing, and then briefly centrifuging:
table 16
1.9.2. The following procedure was set, the 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 beads were added to the sample, and the mixture was gently pipetted and stirred.
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 seconds.
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, extracting corresponding methylation sites by using bismar_methyl_extrator software, and finally calculating the methylation level of each marker.
Example 2
Based on 20 samples clinically diagnosed as cervical cancer collected from Beijing area and 24 healthy human samples collected from Beijing area, biomarkers related to cervical cancer are screened by using methylation level differences among different groups (cervical cancer and control) according to the methylation library building method described in the embodiment 1, and independent data sets of site data in normal and cervical cancer samples are verified, 9 DNA fragments most notably distinguishing normal control from cancer samples are screened out, and the 9 methylation biomarkers (hereinafter referred to as sites or markers) and discrimination thresholds are shown in Table 18.
The methylation level threshold calculation method comprises the following steps: drawing an ROC curve with R-packet pROC from the dataset (containing the type and methylation level of each sample), the confusion matrix corresponding to the optimal threshold point on the ROC curve will be the basis for our calculation of sensitivity (sensitivity), specificity (accuracy) and accuracy. Typically we will choose by a boulder index (you index). The about index, also called the correct index, refers to the sum of sensitivity and specificity minus 1: youden index=sensitivity+specificity-1. The value of about dengue index range is between 0 and 1, which represents the total ability of the classification model to find true patients and non-patients. The greater the about log index, the better the classification model performance, the threshold, sensitivity and specificity of each marker are shown in table 18.
As can be seen from table 18, the AUC values for the markers of the application are higher.
TABLE 18 characterization data for 9 methylation markers
SEQ ID Threshold value Specificity (specificity) Sensitivity of AUC
SEQ ID NO.1 0.300 0.817 0.781 0.85
SEQ ID NO.2 0.195 0.914 0.806 0.89
SEQ ID NO.3 0.146 0.936 0.783 0.91
SEQ ID NO.4 0.133 0.868 0.739 0.82
SEQ ID NO.5 0.165 0.900 0.740 0.88
SEQ ID NO.6 0.133 0.815 0.777 0.91
SEQ ID NO.7 0.129 0.898 0.731 0.86
SEQ ID NO.8 0.122 0.881 0.767 0.94
SEQ ID NO.9 0.285 0.899 0.799 0.86
Example 3
10 human samples (S1-5 is a healthy human sample, S6-10 is a cervical 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 disease condition exceeds the threshold value, the disease condition is a cancer sample, and if the disease condition is lower than the threshold value, the disease condition is a healthy human sample, and the specific results are shown in Table 19:
Wherein, the interpretation result, 0, represents the classification as normal, i.e. healthy; 1 represents a classification as abnormal, i.e. tumor.
Table 19 methylation values and interpretation results for samples
In summary, the inventors of the present invention have obtained a methylated gene related to cervical cancer and determined a target sequence of methylation abnormality of the cervical cancer methylated gene, and through the target sequence of the methylated gene, the methylation state of the gene can be sensitively and specifically detected, so that the methylation state can be used for detecting free DNA of peripheral blood, and the composition of the present invention can realize real-time monitoring, and has higher sensitivity and accuracy.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.
The sequence table is shown in table 20:
table 20
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Sequence listing
<110> Bo' er Cheng (Beijing) technology Co., ltd
<120> markers for cervical cancer screening, probe compositions and uses thereof
<130> PE02003
<160> 81
<170> PatentIn version 3.5
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<211> 60
<212> DNA
<213> artificial sequence
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<223> artificial sequence description: artificially synthesized sequences
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<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 2
tccggcgtct cccctcggcg gttgctttcg ctgccctcaa gagaactcag cttgccggaa 60
gctggttgtt cgctgcggcg accagctccg gaaagcgcgg tggggacgcg ctg 113
<210> 3
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 3
cccctcgccc ccagcagagc agctttccaa cagaaagcaa gtctccccag aaccctccgg 60
<210> 4
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 4
ggggccttag gtaagtgcga ctttggacca cgatacacag acagagcttt gaacgtggct 60
<210> 5
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 5
gaggtcgttc ataaaacaga tggggcaaaa cctgttttgg gagccttccc agtgtccgcc 60
<210> 6
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 6
gccttagata gaacccataa gcctgaaaga attgccatcc ctacaattga cagcgtttct 60
<210> 7
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 7
cgatgcgggc actttgaaga atcacgtgtg ttgaggccct cttaaagaga taggcaccta 60
<210> 8
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 8
ttccacgaag ctgatttaat aagtgctgtc tgtgatgttt ggcaagcctg gtttatatcc 60
<210> 9
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 9
gatccggctc tttgaagttt tcccccaagc ggactcgagg cggcgagaag gagcgggggg 60
<210> 10
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 10
aacgcgcaac tattctcaac tcgcattaaa ccacaaaaaa caaatattac ccgattacta 60
aaaacgttac taaaaaacga taacgcccct ccacaactcg aaaacaactt tcctataaat 120
<210> 11
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 11
acctacaaaa aaattactcc cgaactataa aaaaacgtca tcgcctccta acaacgctcc 60
taacaaccga ataacatcta ctccctataa tccaatacga actaaaaaca actacgcgcc 120
<210> 12
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 12
aacacaacgc gtccccaccg cgctttccga aactaatcgc cgcaacgaac aaccaacttc 60
cgacaaacta aattctctta aaaacaacga aaacaaccgc cgaaaaaaaa cgccgaaaat 120
<210> 13
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 13
acttccgacg tctcccctcg acgattactt tcgctaccct caaaaaaact caacttaccg 60
aaaactaatt attcgctacg acgaccaact ccgaaaaacg cgataaaaac gcgctatatt 120
<210> 14
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 14
aatataaaaa ataaacgacc gccaaacgaa ccgaaaaatt ctaaaaaaac ttactttcta 60
ttaaaaaact actctactaa aaacgaaaaa ccgcccgccc gcccgcccgc caacgctcga 120
<210> 15
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 15
ccgaacgcta acgaacgaac gaacgaacga cccctcgccc ccaacaaaac aactttccaa 60
caaaaaacaa atctccccaa aaccctccga cccgcctaac gaccgcccac tccccatacc 120
<210> 16
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 16
tataacattc ctatctctcc aactaaaaaa aaccacgttc aaaactctat ctatatatcg 60
taatccaaaa tcgcacttac ctaaaacccc ctctaaacac aaaatctctt aaaaccgaaa 120
<210> 17
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 17
ccccgatctt aaaaaatcct atatccaaaa aaaaccttaa ataaatacga ctttaaacca 60
cgatacacaa acaaaacttt aaacgtaact tttcctaact aaaaaaacaa aaatattata 120
<210> 18
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 18
aaaataaata tatctaattt aaaaaacgaa aacgaacact aaaaaaactc ccaaaacaaa 60
ttttacccca tctattttat aaacgacctc ttcaaaccaa aaaccgaatc ccaaaaataa 120
<210> 19
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 19
ccacccctaa aatccgactt ctaacctaaa aaaatcgttc ataaaacaaa taaaacaaaa 60
cctattttaa aaaccttccc aatatccgcc cccgctctcc aaaccaaaca cactcatctc 120
<210> 20
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 20
ttacaaccac atacaccaaa acaacgaccg aaaaacgcta tcaattataa aaataacaat 60
tctttcaaac ttataaattc tatctaaaac tcttaaaatt aaaaaacgcc aatatcacct 120
<210> 21
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 21
aaataatact aacgctcctc aatcccaaaa accttaaata aaacccataa acctaaaaaa 60
attaccatcc ctacaattaa caacgtttct cgaccgttac cctaatacat ataactacaa 120
<210> 22
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 22
tcaaaacgat atttaaaata aaaaaaaaaa taaataccta tctctttaaa aaaacctcaa 60
cacacgtaat tcttcaaaat acccgcatcg accgcccgaa cgcgcaaaac tacttcctac 120
<210> 23
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 23
ataaaaaaca actttacgcg cccgaacgac cgatacgaac actttaaaaa atcacgtata 60
ttaaaaccct cttaaaaaaa taaacaccta cttttccctc caccctaaac accgccctaa 120
<210> 24
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 24
cgacaacata aattcgcaaa ccttataaat aaatataaac caaacttacc aaacatcaca 60
aacaacactt attaaatcaa cttcgtaaaa tacaatacgc ctactatcta atccctccct 120
<210> 25
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 25
aaaaaaaaac taaacaacaa acgtattaca ttccacgaaa ctaatttaat aaatactatc 60
tataatattt aacaaaccta atttatatcc atctacaaaa tttacgaacc catattaccg 120
<210> 26
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 26
ccctcatatc tctctccacc cacccaaccg ccccccgctc cttctcgccg cctcgaatcc 60
gcttaaaaaa aaacttcaaa aaaccgaatc gcaaactccc tacctactcc cccaccgaaa 120
<210> 27
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 27
ccccgataaa aaaataaaca aaaaacctac gatccgactc tttaaaattt tcccccaaac 60
gaactcgaaa cgacgaaaaa aaacgaaaaa cgattaaata aataaaaaaa aatataaaaa 120
<210> 28
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 28
aacacacaac tattctcaac tcacattaaa ccacaaaaaa caaatattac ccaattacta 60
aaaacattac taaaaaacaa taacacccct ccacaactca aaaacaactt tcctataaat 120
<210> 29
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 29
acctacaaaa aaattactcc caaactataa aaaaacatca tcacctccta acaacactcc 60
taacaaccaa ataacatcta ctccctataa tccaatacaa actaaaaaca actacacacc 120
<210> 30
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 30
aacacaacac atccccacca cactttccaa aactaatcac cacaacaaac aaccaacttc 60
caacaaacta aattctctta aaaacaacaa aaacaaccac caaaaaaaaa caccaaaaat 120
<210> 31
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 31
acttccaaca tctcccctca acaattactt tcactaccct caaaaaaact caacttacca 60
aaaactaatt attcactaca acaaccaact ccaaaaaaca caataaaaac acactatatt 120
<210> 32
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 32
aatataaaaa ataaacaacc accaaacaaa ccaaaaaatt ctaaaaaaac ttactttcta 60
ttaaaaaact actctactaa aaacaaaaaa ccacccaccc acccacccac caacactcaa 120
<210> 33
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 33
ccaaacacta acaaacaaac aaacaaacaa cccctcaccc ccaacaaaac aactttccaa 60
caaaaaacaa atctccccaa aaccctccaa cccacctaac aaccacccac tccccatacc 120
<210> 34
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 34
tataacattc ctatctctcc aactaaaaaa aaccacattc aaaactctat ctatatatca 60
taatccaaaa tcacacttac ctaaaacccc ctctaaacac aaaatctctt aaaaccaaaa 120
<210> 35
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 35
ccccaatctt aaaaaatcct atatccaaaa aaaaccttaa ataaatacaa ctttaaacca 60
caatacacaa acaaaacttt aaacataact tttcctaact aaaaaaacaa aaatattata 120
<210> 36
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 36
aaaataaata tatctaattt aaaaaacaaa aacaaacact aaaaaaactc ccaaaacaaa 60
ttttacccca tctattttat aaacaacctc ttcaaaccaa aaaccaaatc ccaaaaataa 120
<210> 37
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 37
ccacccctaa aatccaactt ctaacctaaa aaaatcattc ataaaacaaa taaaacaaaa 60
cctattttaa aaaccttccc aatatccacc cccactctcc aaaccaaaca cactcatctc 120
<210> 38
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 38
ttacaaccac atacaccaaa acaacaacca aaaaacacta tcaattataa aaataacaat 60
tctttcaaac ttataaattc tatctaaaac tcttaaaatt aaaaaacacc aatatcacct 120
<210> 39
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 39
aaataatact aacactcctc aatcccaaaa accttaaata aaacccataa acctaaaaaa 60
attaccatcc ctacaattaa caacatttct caaccattac cctaatacat ataactacaa 120
<210> 40
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 40
tcaaaacaat atttaaaata aaaaaaaaaa taaataccta tctctttaaa aaaacctcaa 60
cacacataat tcttcaaaat acccacatca accacccaaa cacacaaaac tacttcctac 120
<210> 41
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 41
ataaaaaaca actttacaca cccaaacaac caatacaaac actttaaaaa atcacatata 60
ttaaaaccct cttaaaaaaa taaacaccta cttttccctc caccctaaac accaccctaa 120
<210> 42
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 42
caacaacata aattcacaaa ccttataaat aaatataaac caaacttacc aaacatcaca 60
aacaacactt attaaatcaa cttcataaaa tacaatacac ctactatcta atccctccct 120
<210> 43
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 43
aaaaaaaaac taaacaacaa acatattaca ttccacaaaa ctaatttaat aaatactatc 60
tataatattt aacaaaccta atttatatcc atctacaaaa tttacaaacc catattacca 120
<210> 44
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 44
ccctcatatc tctctccacc cacccaacca ccccccactc cttctcacca cctcaaatcc 60
acttaaaaaa aaacttcaaa aaaccaaatc acaaactccc tacctactcc cccaccaaaa 120
<210> 45
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 45
ccccaataaa aaaataaaca aaaaacctac aatccaactc tttaaaattt tcccccaaac 60
aaactcaaaa caacaaaaaa aaacaaaaaa caattaaata aataaaaaaa aatataaaaa 120
<210> 46
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 46
aggggcgtta tcgtttttta gtaacgtttt tagtaatcgg gtaatatttg ttttttgtgg 60
<210> 47
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 47
ttatagggag tagatgttat tcggttgtta ggagcgttgt taggaggcga tgacgttttt 60
<210> 48
<211> 113
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 48
ttcggcgttt tttttcggcg gttgttttcg ttgtttttaa gagaatttag tttgtcggaa 60
gttggttgtt cgttgcggcg attagtttcg gaaagcgcgg tggggacgcg ttg 113
<210> 49
<211> 113
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 49
tagcgcgttt ttatcgcgtt tttcggagtt ggtcgtcgta gcgaataatt agttttcggt 60
aagttgagtt tttttgaggg tagcgaaagt aatcgtcgag gggagacgtc gga 113
<210> 50
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 50
tttttcgttt ttagtagagt agttttttaa tagaaagtaa gtttttttag aatttttcgg 60
<210> 51
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 51
tcggagggtt ttggggagat ttgttttttg ttggaaagtt gttttgttgg gggcgagggg 60
<210> 52
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 52
ggggttttag gtaagtgcga ttttggatta cgatatatag atagagtttt gaacgtggtt 60
<210> 53
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 53
agttacgttt aaagttttgt ttgtgtatcg tggtttaaag tcgtatttat ttaaggtttt 60
<210> 54
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 54
gaggtcgttt ataaaataga tggggtaaaa tttgttttgg gagttttttt agtgttcgtt 60
<210> 55
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 55
ggcggatatt gggaaggttt ttaaaatagg ttttgtttta tttgttttat gaacgatttt 60
<210> 56
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 56
gttttagata gaatttataa gtttgaaaga attgttattt ttataattga tagcgttttt 60
<210> 57
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 57
agaaacgttg ttaattgtag ggatggtaat ttttttaggt ttatgggttt tatttaaggt 60
<210> 58
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 58
cgatgcgggt attttgaaga attacgtgtg ttgaggtttt tttaaagaga taggtattta 60
<210> 59
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 59
taggtgttta tttttttaag agggttttaa tatacgtgat tttttaaagt gttcgtatcg 60
<210> 60
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 60
ttttacgaag ttgatttaat aagtgttgtt tgtgatgttt ggtaagtttg gtttatattt 60
<210> 61
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 61
ggatataaat taggtttgtt aaatattata gatagtattt attaaattag tttcgtggaa 60
<210> 62
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 62
gattcggttt tttgaagttt ttttttaagc ggattcgagg cggcgagaag gagcgggggg 60
<210> 63
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 63
tttttcgttt tttttcgtcg tttcgagttc gtttggggga aaattttaaa gagtcggatt 60
<210> 64
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 64
aggggtgtta ttgtttttta gtaatgtttt tagtaattgg gtaatatttg ttttttgtgg 60
<210> 65
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 65
ttatagggag tagatgttat ttggttgtta ggagtgttgt taggaggtga tgatgttttt 60
<210> 66
<211> 113
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 66
tttggtgttt ttttttggtg gttgtttttg ttgtttttaa gagaatttag tttgttggaa 60
gttggttgtt tgttgtggtg attagttttg gaaagtgtgg tggggatgtg ttg 113
<210> 67
<211> 113
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 67
tagtgtgttt ttattgtgtt ttttggagtt ggttgttgta gtgaataatt agtttttggt 60
aagttgagtt tttttgaggg tagtgaaagt aattgttgag gggagatgtt gga 113
<210> 68
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 68
ttttttgttt ttagtagagt agttttttaa tagaaagtaa gtttttttag aattttttgg 60
<210> 69
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 69
ttggagggtt ttggggagat ttgttttttg ttggaaagtt gttttgttgg gggtgagggg 60
<210> 70
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 70
ggggttttag gtaagtgtga ttttggatta tgatatatag atagagtttt gaatgtggtt 60
<210> 71
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 71
agttatgttt aaagttttgt ttgtgtattg tggtttaaag ttgtatttat ttaaggtttt 60
<210> 72
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 72
gaggttgttt ataaaataga tggggtaaaa tttgttttgg gagttttttt agtgtttgtt 60
<210> 73
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 73
ggtggatatt gggaaggttt ttaaaatagg ttttgtttta tttgttttat gaatgatttt 60
<210> 74
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 74
gttttagata gaatttataa gtttgaaaga attgttattt ttataattga tagtgttttt 60
<210> 75
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 75
agaaatgttg ttaattgtag ggatggtaat ttttttaggt ttatgggttt tatttaaggt 60
<210> 76
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 76
tgatgtgggt attttgaaga attatgtgtg ttgaggtttt tttaaagaga taggtattta 60
<210> 77
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 77
taggtgttta tttttttaag agggttttaa tatatgtgat tttttaaagt gtttgtattg 60
<210> 78
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 78
ttttatgaag ttgatttaat aagtgttgtt tgtgatgttt ggtaagtttg gtttatattt 60
<210> 79
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 79
ggatataaat taggtttgtt aaatattata gatagtattt attaaattag ttttgtggaa 60
<210> 80
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 80
gatttggttt tttgaagttt ttttttaagt ggatttgagg tggtgagaag gagtgggggg 60
<210> 81
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence description: artificially synthesized sequences
<400> 81
ttttttgttt ttttttgttg ttttgagttt gtttggggga aaattttaaa gagttggatt 60

Claims (8)

1. The application of the marker in the preparation of a kit for detecting cervical cancer is characterized in that the marker is a LINC00643 gene, the nucleotide sequence of the LINC00643 gene is shown as SEQ ID NO. 1, and the marker is a methylated marker.
2. The use according to claim 1, wherein the marker further comprises one of the following genes: ZNF773, CITED1, GABRA1, BCOR, NXPH1, DIAPH2, FAM133A, and BCOR.
3. A composition for cervical cancer detection, characterized in that the composition comprises a nucleic acid for detecting methylation of the LINC00643 gene,
the nucleotide sequence of the LINC00643 gene is shown as SEQ ID NO. 1,
the nucleic acid comprises a probe composition for targeting the methylated LINC00643 gene of cervical cancer;
The probe composition comprising a hypermethylated first probe composition for hybridization with a region of high methylation of the bisulfite converted CG and a hypomethylated second probe composition for hybridization with a region of low methylation of the bisulfite converted CG,
the first probe composition comprises a nucleotide sequence shown as SEQ ID NO. 10-11; the second probe composition comprises the nucleotide sequence shown as SEQ ID NOS.28-29.
4. The composition of claim 3, further comprising a nucleic acid for detecting methylation of one of the following genes: ZNF773, CITED1, GABRA1, BCOR, NXPH1, DIAPH2, FAM133A, and BCOR.
5. The composition of claim 3 or 4, further comprising an agent that converts an unmethylated cytosine base at position 5 of the target sequence of the marker to uracil.
6. The composition of claim 3 or 4, wherein the nucleic acid for detecting methylation of a target sequence of a marker further comprises:
blocking agents that preferentially bind to target sequences in the unmethylated state.
7. A kit comprising the composition of any one of claims 3-6.
8. A chip comprising the composition of any one of claims 3-6.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111808962A (en) * 2020-07-23 2020-10-23 深圳市新合生物医疗科技有限公司 Kit for cervical cancer detection and use method
CN113444804A (en) * 2021-07-14 2021-09-28 武汉大学中南医院 Cervical cancer prognosis related gene and application thereof in preparation of cervical cancer prognosis prediction and diagnosis product

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KR101374822B1 (en) * 2010-04-23 2014-03-17 (주)지노믹트리 Methylation Marker For Cervical Cancer Diagnosis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111808962A (en) * 2020-07-23 2020-10-23 深圳市新合生物医疗科技有限公司 Kit for cervical cancer detection and use method
CN113444804A (en) * 2021-07-14 2021-09-28 武汉大学中南医院 Cervical cancer prognosis related gene and application thereof in preparation of cervical cancer prognosis prediction and diagnosis product

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