CN114540497B - Marker for bladder cancer screening, probe composition and application thereof - Google Patents

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

Info

Publication number
CN114540497B
CN114540497B CN202210182495.9A CN202210182495A CN114540497B CN 114540497 B CN114540497 B CN 114540497B CN 202210182495 A CN202210182495 A CN 202210182495A CN 114540497 B CN114540497 B CN 114540497B
Authority
CN
China
Prior art keywords
seq
marker
methylation
composition
probe
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210182495.9A
Other languages
Chinese (zh)
Other versions
CN114540497A (en
Inventor
连明
韩晓亮
王小奇
刘栓平
魏闯
张聪
杨亚东
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biochain Beijing Science and Technology Inc
Original Assignee
Biochain Beijing Science and Technology Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biochain Beijing Science and Technology Inc filed Critical Biochain Beijing Science and Technology Inc
Priority to CN202210182495.9A priority Critical patent/CN114540497B/en
Publication of CN114540497A publication Critical patent/CN114540497A/en
Application granted granted Critical
Publication of CN114540497B publication Critical patent/CN114540497B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Hospice & Palliative Care (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The application discloses a marker for bladder cancer screening, a probe composition and application thereof, wherein the marker is selected from any one of 3 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 can be 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 bladder cancer screening, probe composition and application thereof
Technical Field
The application relates to the field of biotechnology, in particular to a marker for bladder cancer screening, a probe composition and application thereof.
Background
Bladder is an important component of the human urinary system, bladder cancer is one of the most frequently occurring malignant tumors of the male genitourinary system, and its morbidity and mortality are in the first place according to global cancer report by WHO in 2020, and in male cancers, the morbidity and mortality are ranked as 6 th (4.4%) and 10 th (2.9%) respectively.
Because the symptoms are obvious, most bladder cancers can be diagnosed early, and most of the diagnosed bladder cancers belong to superficial non-invasive bladder cancers, wherein most of the bladder cancers are stage 0 and stage I, and the bladder cancers can achieve a very good effect only by using a scheme mainly used for surgery. In general, the 5-year survival rate of stage 0 bladder cancer is as high as 98%, stage I is also nearly 90%, and most of the bladder cancer can be cured. However, about 70% of bladder cancers recur within 3 years after the first surgery, so how to prevent tumor recurrence is an important component of effective treatment.
Early findings are critical to improving patient survival and cure rate, but currently lack accurate non-invasive diagnostic methods. 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 object of the present application is to provide a marker for detecting bladder cancer, which can be used for screening bladder cancer, wherein the marker is used for screening asymptomatic people in a non-invasive way, and for 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 application is as follows:
1. a marker for detecting bladder cancer, the marker selected from one of: ptpur, SEPTIN9 or CRYBG1.
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 NOs 1 to 3, preferably the marker is a methylated marker.
3. A probe composition comprising a probe that targets methylation of the marker of item 1 or 2.
4. The probe composition of item 3, comprising a hypermethylated first probe composition for hybridization to a bisulfite converted hypermethylated region and a hypomethylated second probe composition for hybridization to a bisulfite converted 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 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 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 of SEQ ID NOS: 4-9 and the second probe composition comprises one or two of SEQ ID NOS: 10-15.
5. Use of a marker selected from one of the following in the manufacture of a kit for detecting bladder cancer: ptpur, SEPTIN9 or CRYBG1.
6. The use according to item 5, wherein the nucleotide sequence of the marker is selected from one of the markers shown in SEQ ID NOs 1 to 3, preferably the marker is a methylated marker;
preferably, the probe composition is used to target a post-methylation marker of bladder cancer;
preferably, the probe composition is the probe composition according to item 3 or 4.
7. A composition for use in the detection of bladder cancer, the composition comprising a nucleic acid for detecting methylation of any one of the markers selected from the group consisting of: ptpur, SEPTIN9 or CRYBG1.
8. The composition of item 7, wherein the nucleotide sequence of the marker is selected from one of the nucleotide sequences shown in SEQ ID NOS.1-3.
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 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 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 present application
The inventor of the present application has found a plurality of methylation genes related to bladder cancer by analyzing genome methylation data of bladder cancer using epigenomic and bioinformatics techniques, and has determined a target sequence of methylation abnormality of the methylation genes of bladder cancer, and can sensitively and specifically detect the methylation state of the genes by the target sequence of the methylation genes, thereby being useful for detecting free DNA of peripheral blood.
The composition is used for screening asymptomatic people in a non-invasive mode, harm caused by invasive detection is reduced, and the composition has higher sensitivity and accuracy and can realize real-time monitoring.
Detailed Description
The present application is described in detail below. While specific embodiments of the present application are shown, it should be understood that the present application 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 disclosure 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 the preferred embodiment for carrying out the present application, but is not intended to limit the scope of the present application in general, as the description proceeds. The scope of the present application is defined by the appended claims.
The present application provides a marker for detecting bladder cancer, the marker being selected from one of the following: ptpur, SEPTIN9 or CRYBG1.
In one embodiment, the nucleotide sequence of the marker is selected from one of the markers shown in SEQ ID NOs 1-3, preferably the marker is a methylated marker.
Wherein the nucleotide sequence of the PTPRU is shown as SEQ ID NO. 1; the nucleotide sequence of SEPTIN9 is shown as SEQ ID NO. 2; the nucleotide sequence of CRYBG1 is shown in SEQ ID NO. 3;
The sequences of the markers are all sequences which are not converted by bisulfite.
The present application 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 as described herein can be methylation level, degree of methylation, or methylation state, and when analyzing methylation of such target sequences, one skilled in the art can 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 this application, the region that complementarily binds or hybridizes to a 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 hypermethylated first probe composition for hybridization to a bisulfite converted hypermethylated region and a hypomethylated second probe composition for hybridization to a bisulfite converted hypomethylated region.
The hypermethylation means that after the marker is converted by bisulfite, a base C is changed into a base U, 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 U.
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. 16;
the complementary strand of the sequence in the extreme case is shown in SEQ ID NO. 17;
the sequence of one extreme case of SEQ ID NO. 2 is shown as SEQ ID NO. 18;
The complementary strand of the sequence in the extreme case is shown in SEQ ID NO. 19;
the sequence of one extreme case of SEQ ID NO. 3 is shown as SEQ ID NO. 20;
the complementary strand of the sequence in the extreme case is shown in SEQ ID NO. 21;
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. 22;
the complementary strand of the sequence in the extreme case is shown in SEQ ID NO. 23;
the sequence of one extreme case of SEQ ID NO. 2 is shown as SEQ ID NO. 24;
the complementary strand of the sequence in the extreme case is shown in SEQ ID NO. 25;
the sequence of one extreme case of SEQ ID NO. 3 is shown as SEQ ID NO. 26;
the complement of the sequence in the extreme case is shown as SEQ ID NO. 27;
in one embodiment, 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 hypermethylated region.
The second probe composition includes m probes that hybridize to each nucleotide of the sense strand and/or the antisense strand of the bisulfite converted 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 one 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 is 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.
The probe composition is hybridized with the marker converted by the bisulfite, wherein the high-methylation first probe composition is hybridized with a high-methylation region, and the low-methylation second probe composition is hybridized with a low-methylation region, so that the methylation level of a target sequence can be detected efficiently and accurately, and the probe composition can be used for bladder cancer screening.
In one embodiment, the hypermethylated first probe composition comprises one or both of SEQ ID NOS: 4-9.
The hypomethylated second probe composition comprises one or two of SEQ ID NOS 10-15.
Wherein the first probe composition for hybridization with a PtPRU methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS.4-5;
the first probe composition for hybridization with the SEPTIN9 methylation sequence comprises the nucleotide sequence shown in SEQ ID NOS.6-7;
the first probe composition for hybridization with CRYBG1 methylation sequences comprises the nucleotide sequences shown in SEQ ID NOS.8-9.
A second probe composition for hybridization with a PtPRU methylation sequence comprises a nucleotide sequence as set forth in SEQ ID NOS 10-11;
a second probe composition for hybridization to a SEPTIN9 methylation sequence comprises the nucleotide sequence shown in SEQ ID NOS.12-13;
a second probe composition for hybridization with CRYBG1 methylation sequences comprises the nucleotide sequences shown in SEQ ID NOS.14-15.
The application provides the use of a marker in the preparation of a kit for detecting bladder cancer, the marker being selected from one of the following: ptpur, SEPTIN9 or CRYBG1.
In one embodiment, the nucleotide sequence of the marker is selected from one of the markers shown in SEQ ID NOs 1-3, preferably the marker is a methylated marker.
The application provides the use of a probe composition for targeting a marker after methylation of bladder cancer in the preparation of a kit for detecting bladder cancer.
In one embodiment, the probe composition is the probe composition described above.
The present application provides a composition for detection of bladder cancer, the composition comprising a nucleic acid for detecting methylation of any one of the markers selected from the group consisting of: PTPRU, SEPTIN9 or CRYBG1, preferably, the nucleotide sequence of the marker is selected from one of the sequences shown in SEQ ID NO. 1-3.
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 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 one 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 one 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 present application provides a kit comprising the above-described marker or the above-described probe composition or the above-described 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 application is not limited in any way with respect to the method for detecting the methylation level of a target sequence using the above-described kit, and one skilled in the art can select as desired, for example, the present application provides a method for detecting the methylation level of a marker target sequence using the above-described kit, which comprises 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 application provides a method for detecting the methylation level of a target sequence of a marker using the kit described above, 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 present application provides a chip comprising the above-described marker or the above-described probe composition or the above-described 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 present application provides a method of screening for bladder cancer comprising:
detecting methylation level of a marker
Determining the risk of the subject to suffer from bladder cancer based on the methylation level, the marker being selected from one of:
ptpur, SEPTIN9 or CRYBG1.
Examples
The materials used in the test and the test methods are generally and/or specifically described herein, and 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), bladder tissue 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 (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, the markers specific to bladder cancer were selected to obtain 93 markers. Meanwhile, the difference between the methylation level of the 450k chip bladder cancer tissue (660) and the methylation level of the paracancerous tissue (210) in TCGA is more than 0.2, and finally 31 differential methylation areas are obtained.
4) And (3) marker verification: the probe capture is designed for the 31 different methylation areas, and the data of the Bohr hong plasma samples (the number of bladder cancer samples=33 and the number of healthy human samples=38) are utilized for verification, so that 3 markers capable of distinguishing bladder cancer from healthy human are finally obtained, and the sequences of the markers are respectively shown as SEQ ID NO: 1-3.
Based on the resulting target sequence region, a probe composition (panel) is tailored comprising a hypermethylated first probe composition and a hypomethylated second probe composition, wherein, for each marker, the first probe composition comprises two probes,
For SEQ ID NO. 1, the first probe composition comprises a nucleotide sequence as shown in SEQ ID NO. 4-5;
for SEQ ID NO. 2, the first probe composition comprises the nucleotide sequence shown as SEQ ID NO. 6-7;
for SEQ ID NO. 3, the first probe composition comprises the nucleotide sequences as shown in SEQ ID NO. 8-9;
the second probe composition comprises two probes, wherein,
for SEQ ID NO. 1, the second probe composition comprises the nucleotide sequence shown as SEQ ID NO. 10-11;
for SEQ ID NO. 2, the second probe composition comprises the nucleotide sequence shown as SEQ ID NO. 12-13;
for SEQ ID NO. 3, the second probe composition comprises the nucleotide sequences as shown in SEQ ID NO. 14-15.
Based on the obtained markers, the probe composition (panel) was tailored.
Example 2 sample preparation, library construction and validation methods
For 1 marker obtained in example 1, which can distinguish bladder cancer from healthy people, the verification was performed in plasma samples, and the specific 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
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
Fragmented DNA per 50 μ lER and AT reaction Concentration of the linker
1μg 10μM
500ng 10μM
250ng 10μM
100ng 10μM
50ng 10μM
25ng 10μM
10ng 3μM
5ng 5μM
2.5ng 2.5μM
1ng 625nM
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 (AgencourtAMPure 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
AgenecurtAMPureXP beads 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 lAgencourtAMPure 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 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. lAgencourtAMPure 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 bismark_methyl_extraction software, and finally calculating the methylation level of each marker, wherein if the value exceeds a threshold value, judging the result as cancer, and if the value is lower than the threshold value, judging the result as normal.
Example 3 determination of methylation threshold
Calculating methylation levels of the 3 methylation biomarkers screened based on 33 samples clinically diagnosed as bladder cancer collected from Beijing area and 38 healthy human samples collected from Beijing area by using the methylation library building method described in example 2, and calculating threshold values (hereinafter referred to as loci or markers) and AUC values of independent distinction according to the methylation levels of the 3 methylation biomarkers in the bladder cancer sample and normal human sample data set as 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 jordrinier index (youden 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 larger 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 described herein are higher.
TABLE 18 characterization data for 3 methylation markers
SEQ ID Threshold value Specificity Sensitivity AUC
SEQ ID NO.1 0.356 0.91 0.89 0.90
SEQ ID NO.2 0.239 0.89 0.94 0.91
SEQ ID NO.3 0.118 0.83 1.00 0.87
Example 4 validation of markers Using clinical samples
Collecting peripheral blood according to the method of example 2 by using a methylation marker detection method of the present application by additionally collecting 6 human samples (S1-3 is a healthy human sample and S4-6 is a bladder cancer patient sample) from Beijing area; 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 application have obtained a methylation gene associated with bladder cancer, and determined a target sequence of methylation abnormality of the methylation gene of bladder cancer, and, through the target sequence of the methylation 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 described in the present application can realize real-time monitoring, with higher sensitivity and accuracy.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application 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 application still fall within the protection scope of the technical solution of the present application.
The sequence table used in the application is shown in table 20:
table 20
/>
/>
Sequence listing
<110> Bo' er Cheng (Beijing) technology Co., ltd
<120> markers for bladder cancer screening, probe compositions and uses thereof
<130> PF02110
<160> 27
<170> PatentIn version 3.5
<210> 1
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 1
ggcgcgggcg tctctaactt cgcggagctc atcgtcaagg gtcagctggt ggacgccggg 60
<210> 2
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 2
gctgctgggg tgagtcctgc tcctttgttc ttcccagcct tgcaccactg gctcgggggc 60
<210> 3
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 3
actcctccgt gttcacatgg taggaactcc cacaggcaaa agcctttccg atgcgctctg 60
<210> 4
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 4
ccgcccctcg aaaataaaat cccgacgctc cccgacgtcc accaactaac ccttaacgat 60
aaactccgcg aaattaaaaa cgcccgcgcc gcgcgaaacc taaaacacac aacgatacaa 120
<210> 5
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 5
ctataccgct atatatccca aaccccgcgc gacgcgaacg tctctaactt cgcgaaactc 60
atcgtcaaaa atcaactaat aaacgccgaa aaacgccgaa acctcaccct cgaaaaacga 120
<210> 6
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 6
aaatccgcct cgcgaccgcg ccacctaaaa acccccgaac caataataca aaactaaaaa 60
aaacaaaaaa acaaaactca ccccaacaac gaaacgtcga actacgctaa acgcgccgaa 120
<210> 7
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 7
ctcgacgcgt ccaacgcaac ccgacgttcc gctactaaaa taaatcctac tcctttattc 60
ttcccaacct tacaccacta actcgaaaac tctcaaataa cgcgaccgcg aaacgaaccc 120
<210> 8
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 8
cgttcaaaca cccgaataat cccgcgacta caaaacgcat cgaaaaaact tttacctata 60
aaaattccta ccatataaac acgaaaaaat acgtaaaaat aacgaacgaa attacaaaaa 120
<210> 9
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 9
cccctacaac ctcgcccgct acccccacgc actcctccgt attcacataa taaaaactcc 60
cacaaacaaa aacctttccg atacgctcta caaccgcgaa atcactcgaa tatttaaacg 120
<210> 10
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 10
ccacccctca aaaataaaat cccaacactc cccaacatcc accaactaac ccttaacaat 60
aaactccaca aaattaaaaa cacccacacc acacaaaacc taaaacacac aacaatacaa 120
<210> 11
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 11
ctataccact atatatccca aaccccacac aacacaaaca tctctaactt cacaaaactc 60
atcatcaaaa atcaactaat aaacaccaaa aaacaccaaa acctcaccct caaaaaacaa 120
<210> 12
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 12
aaatccacct cacaaccaca ccacctaaaa acccccaaac caataataca aaactaaaaa 60
aaacaaaaaa acaaaactca ccccaacaac aaaacatcaa actacactaa acacaccaaa 120
<210> 13
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 13
ctcaacacat ccaacacaac ccaacattcc actactaaaa taaatcctac tcctttattc 60
ttcccaacct tacaccacta actcaaaaac tctcaaataa cacaaccaca aaacaaaccc 120
<210> 14
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 14
cattcaaaca cccaaataat cccacaacta caaaacacat caaaaaaact tttacctata 60
aaaattccta ccatataaac acaaaaaaat acataaaaat aacaaacaaa attacaaaaa 120
<210> 15
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 15
cccctacaac ctcacccact acccccacac actcctccat attcacataa taaaaactcc 60
cacaaacaaa aacctttcca atacactcta caaccacaaa atcactcaaa tatttaaaca 120
<210> 16
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 16
ggcgcgggcg tttttaattt cgcggagttt atcgttaagg gttagttggt ggacgtcggg 60
<210> 17
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 17
ttcggcgttt attagttgat ttttgacgat gagtttcgcg aagttagaga cgttcgcgtt 60
<210> 18
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 18
gttgttgggg tgagttttgt ttttttgttt tttttagttt tgtattattg gttcgggggt 60
<210> 19
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 19
gttttcgagt tagtggtgta aggttgggaa gaataaagga gtaggattta ttttagtagt 60
<210> 20
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 20
attttttcgt gtttatatgg taggaatttt tataggtaaa agttttttcg atgcgttttg 60
<210> 21
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 21
tagagcgtat cggaaaggtt tttgtttgtg ggagttttta ttatgtgaat acggaggagt 60
<210> 22
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 22
ggtgtgggtg tttttaattt tgtggagttt attgttaagg gttagttggt ggatgttggg 60
<210> 23
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 23
tttggtgttt attagttgat ttttgatgat gagttttgtg aagttagaga tgtttgtgtt 60
<210> 24
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 24
gttgttgggg tgagttttgt ttttttgttt tttttagttt tgtattattg gtttgggggt 60
<210> 25
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 25
gtttttgagt tagtggtgta aggttgggaa gaataaagga gtaggattta ttttagtagt 60
<210> 26
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 26
atttttttgt gtttatatgg taggaatttt tataggtaaa agtttttttg atgtgttttg 60
<210> 27
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: artificially synthesized sequences
<400> 27
tagagtgtat tggaaaggtt tttgtttgtg ggagttttta ttatgtgaat atggaggagt 60

Claims (9)

1. Use of a methylated marker in the manufacture of a kit for detecting bladder cancer, characterized in that the methylated marker is a methylated PTPRU having the nucleotide sequence of SEQ ID No. 1;
bladder cancer is screened by measuring the level of PTPRU methylation in the test sample.
2. The use according to claim 1, wherein the methylation marker further comprises methylated SEPTIN9 and/or methylated CRYBG1; wherein,
the nucleotide sequence of SEPTIN9 is SEQ ID NO. 2;
the nucleotide sequence of CRYBG1 is SEQ ID NO 3.
3. A composition for bladder cancer detection, comprising a nucleic acid for detecting a methylation marker, wherein the methylation marker is a methylated PTPRU, and wherein the nucleotide sequence of the PTPRU is SEQ ID No. 1;
The composition further comprises an agent that converts an unmethylated cytosine base at position 5 of the target sequence of the marker to uracil;
the nucleic acid comprises: a probe composition for detecting a methylation marker;
the probe composition includes a hypermethylated first probe composition for hybridization with a bisulfite converted hypermethylated region and a hypomethylated second probe composition for hybridization with a bisulfite converted hypomethylated region.
4. The composition of claim 3, wherein the methylation marker further comprises methylated SEPTIN9 and/or methylated CRYBG1; wherein,
the nucleotide sequence of SEPTIN9 is SEQ ID NO. 2;
the nucleotide sequence of CRYBG1 is SEQ ID NO 3.
5. The composition of claim 4, wherein,
for SEQ ID NO. 1, the first probe composition comprises a nucleotide sequence as shown in SEQ ID NO. 4-5;
for SEQ ID NO. 2, the first probe composition comprises the nucleotide sequence shown as SEQ ID NO. 6-7;
for SEQ ID NO. 3, the first probe composition comprises the nucleotide sequences as shown in SEQ ID NO. 8-9.
6. The composition of claim 4, wherein for SEQ ID NO. 1, the second probe composition comprises a nucleotide sequence as set forth in SEQ ID NO. 10-11;
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 sequences as shown in SEQ ID NO. 14-15.
7. The composition of claim 3, wherein the nucleic acid for detecting methylation of a target sequence of a marker further comprises:
a blocking agent that binds to a target sequence in an unmethylated state.
8. A kit comprising the composition of any one of claims 3-7.
9. A chip comprising the composition of any one of claims 3-7.
CN202210182495.9A 2022-02-25 2022-02-25 Marker for bladder cancer screening, probe composition and application thereof Active CN114540497B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210182495.9A CN114540497B (en) 2022-02-25 2022-02-25 Marker for bladder cancer screening, probe composition and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210182495.9A CN114540497B (en) 2022-02-25 2022-02-25 Marker for bladder cancer screening, probe composition and application thereof

Publications (2)

Publication Number Publication Date
CN114540497A CN114540497A (en) 2022-05-27
CN114540497B true CN114540497B (en) 2024-02-27

Family

ID=81678736

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210182495.9A Active CN114540497B (en) 2022-02-25 2022-02-25 Marker for bladder cancer screening, probe composition and application thereof

Country Status (1)

Country Link
CN (1) CN114540497B (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107727865A (en) * 2016-08-11 2018-02-23 博尔诚（北京）科技有限公司 The systemic detection method of tumor markers and its application
CN107723363A (en) * 2016-08-11 2018-02-23 博尔诚（北京）科技有限公司 The combined detection method of tumor markers and its application
CN111676286A (en) * 2020-05-29 2020-09-18 武汉爱基百客生物科技有限公司 Multiplex PCR primer system for detecting free DNA methylation of lung cancer plasma, detection method and application
CN112662760A (en) * 2020-02-25 2021-04-16 博尔诚（北京）科技有限公司 Cancer gene methylation detection system and cancer in-vitro detection method implemented in cancer gene methylation detection system

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102796808B (en) * 2011-05-23 2014-06-18 深圳华大基因科技服务有限公司 Methylation high-flux detection method
WO2016205236A1 (en) * 2015-06-15 2016-12-22 The Johns Hopkins University Methods of detection and treatment of urothelial cancer

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107727865A (en) * 2016-08-11 2018-02-23 博尔诚（北京）科技有限公司 The systemic detection method of tumor markers and its application
CN107723363A (en) * 2016-08-11 2018-02-23 博尔诚（北京）科技有限公司 The combined detection method of tumor markers and its application
CN112662760A (en) * 2020-02-25 2021-04-16 博尔诚（北京）科技有限公司 Cancer gene methylation detection system and cancer in-vitro detection method implemented in cancer gene methylation detection system
WO2021169875A1 (en) * 2020-02-25 2021-09-02 博尔诚（北京）科技有限公司 Cancer gene methylation measuring system and cancer in vitro detection method executed in same
CN111676286A (en) * 2020-05-29 2020-09-18 武汉爱基百客生物科技有限公司 Multiplex PCR primer system for detecting free DNA methylation of lung cancer plasma, detection method and application

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Aberrant Promoter Methylation of Multiple Genes during Pathogenesis of Bladder Cancer;Mariana Brait等;Cancer Epidemiol Biomarkers Prev;第17卷(第10期);全文 *
Jean-Pierre Roperch等.Promoter hypermethylation of HS3ST2,SEPTIN9 and SLIT2 combined with FGFR3 mutations as a sensitive/specific urinary assay for diagnosis and surveillance in patients with low or high-risk non-muscleinvasive bladder cancer.Roperch et al. BMC Cancer.2016,第16卷(第1期),参见对比文件1摘要、表3及其相关注释. *
Promoter hypermethylation of HS3ST2,SEPTIN9 and SLIT2 combined with FGFR3 mutations as a sensitive/specific urinary assay for diagnosis and surveillance in patients with low or high-risk non-muscleinvasive bladder cancer;Jean-Pierre Roperch等;Roperch et al. BMC Cancer;第16卷(第1期);参见对比文件1摘要、表3及其相关注释 *

Also Published As

Publication number Publication date
CN114540497A (en) 2022-05-27

Similar Documents

Publication Publication Date Title
WO2021169875A1 (en) Cancer gene methylation measuring system and cancer in vitro detection method executed in same
WO2021180106A1 (en) Probe composition for detecting five tumors of digestive tract
WO2021185274A1 (en) Probe composition for detecting 6 cancers with high incidence in china
CN118272525A (en) Marker for gastric cancer screening, probe composition and application thereof
WO2021180105A1 (en) Probe composition for detecting common cancers of both sexes
WO2021169874A1 (en) Probe composition for detecting three lumen organ tumors
WO2021175284A1 (en) Probe composition for detecting three types of solid organ tumors
CN114540497B (en) Marker for bladder cancer screening, probe composition and application thereof
CN114507734B (en) Marker for thyroid cancer screening, probe composition and application thereof
CN114369664B (en) Marker for pancreatic cancer screening, probe composition and application thereof
CN114703281B (en) Marker for screening testicular cancer, probe composition and application thereof
CN114410792B (en) Marker for kidney cancer screening, probe composition and application thereof
CN114317732B (en) Composition for lung cancer screening and application thereof
CN114231635B (en) Marker and probe composition for lung cancer screening and application thereof
CN115279924A (en) Probe composition for detecting 11 cancers
CN114395626B (en) Marker for cervical cancer screening, probe composition and application thereof
CN114395628B (en) Marker for colorectal cancer screening, probe composition and application thereof
CN114369663A (en) Marker and probe composition for liver cancer screening and application thereof
CN118048451A (en) Marker for screening liver cancer, probe composition and application thereof
CN114369661A (en) Marker and probe composition for breast cancer screening and application thereof
CN117448450A (en) Marker for colorectal cancer screening, probe composition and application thereof
CN117025777A (en) Marker for assessing thyroid cancer risk, probe composition, system and application thereof
CN117025776A (en) Marker for assessing thyroid cancer risk, probe composition, system and application thereof
CN116024342A (en) Nucleic acid product for detecting cervical cancer or precancerous lesions, kit and application
CN117625782A (en) Marker for early screening of lung cancer, probe composition and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant