Marker and probe composition for detecting endometrial cancer and application of marker and probe composition

Abstract

The invention discloses a marker and a probe composition for detecting endometrial cancer and application of the marker and the probe composition. The marker is a TBX15 gene or an MAP10 gene. The marker can sensitively and specifically detect the endometrial cancer and can be used for screening the endometrial cancer, 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 be used for detecting the endometrial cancer. And real-time monitoring can be realized.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a marker and a probe composition for detecting endometrial cancer and applications thereof. Background Art

[0002] Endometrial cancer is an epithelial malignant tumor that occurs in the endometrium. It is more common in postmenopausal women and has a tendency to become younger in recent years. Most endometrial cancers are localized lesions, and the opportunity for early diagnosis is often lost due to ignoring early symptoms. Studies have shown that young, early, and low-grade endometrial cancer patients have a better survival rate.

[0003] Early detection and diagnosis of endometrial cancer mainly include imaging and histopathological screening: (1) Transvaginal ultrasound is the most commonly used imaging examination, and the main symptoms are endometrial thickening, disappearance of the uterine cavity line, and uneven intrauterine echo. (2) CT and MRI also play an important role in the diagnosis of endometrial cancer. Their performance in judging myometrial invasion or lymph node metastasis of endometrial cancer before surgery is far better than that of ultrasound diagnosis. (3) Histopathological examination mainly includes diagnostic curettage and hysteroscopic biopsy plus diagnostic surgery. However, the application scope of diagnostic curettage is relatively limited and the missed diagnosis rate is high. Endometrial biopsy under hysteroscopy is more intuitive and is the gold standard for diagnosing endometrial lesions. (4) Endometrial cytology screening is also an important means of diagnosing endometrial cancer, but it cannot be used as a basis for diagnosis. It has the advantages of high satisfaction with the sample, less pain, and less trauma. (5) Tumor markers such as cancer antigen 125 (CA125) and human epididymis protein 4 (HE4) are mostly in the clinical stage of diagnosis of endometrial cancer and cannot be used as screening or diagnosis methods.

[0004] At present, the clinical commonly used endometrial cell collector combined with liquid-based cytology preparation technology for early detection of endometrial cancer, the sensitivity of diagnosing atypical endometrial hyperplasia is 55%, the specificity is 82.6%; the sensitivity of diagnosing endometrial cancer is 36.4%, the specificity is 99.7%. The use of endometrial cell collectors to collect samples and perform liquid-based cytology examinations can be used to screen endometrial lesions in symptomatic people and high-risk groups, but the diagnostic technology has poor sensitivity. Therefore, there is currently a lack of effective non-invasive screening methods for endometrial cancer in the general population. As the incidence of endometrial cancer in China shows an increasing trend every year, the exploration of new screening methods is urgent. Summary of the invention

[0005] The purpose of the present application is to provide a marker, a probe composition and an application for detecting endometrial cancer, which can be used for early screening of endometrial cancer. The marker is used in a non-invasive manner for screening of asymptomatic people and prognosis detection of cancer patients, reducing the harm caused by invasive detection and having higher sensitivity and accuracy.

[0006] The specific technical solutions of this application are as follows:

[0007] 1. A marker for detecting endometrial cancer, wherein the marker is the TBX15 gene or the MAP10 gene.

[0008] 2. The marker according to item 1, wherein the target sequence of the TBX15 gene is as shown in any one of SEQ ID NOs: 1-6, or includes the sequence shown in any one of SEQ ID NOs: 1-6; the target sequence of the MAP10 gene is as shown in any one of SEQ ID NOs: 7-12, or includes the sequence shown in any one of SEQ ID NOs: 7-12.

[0009] 3. A probe composition, wherein the probe composition comprises a probe targeting the methylation of the marker described in item 1 or 2.

[0010] 4. The probe composition according to item 3, wherein the probe composition comprises a first probe composition with high methylation and a second probe composition with low methylation, the first probe composition is used to hybridize with a region with high methylation of CG converted by bisulfite, and the second probe composition is used to hybridize with a region with low methylation of CG converted by bisulfite.

[0011] 5. The probe composition according to item 4, wherein the first probe composition comprises n probes, and the n probes hybridize with each nucleotide of the sense chain and / or antisense chain of the CG high methylation region converted by bisulfite. Preferably, the second probe composition comprises m probes, and the m probes hybridize with each nucleotide of the sense chain and / or antisense chain of the CG low methylation region converted by bisulfite. Further preferably, n and m are any integers in the range of 1-10.

[0012] 6. The probe composition according to item 5, wherein there is x1 nucleotide overlap between the n-1th probe and the nth probe, and preferably, x1 is any integer between 0 and 100;

[0013] Preferably, there are x2 nucleotides overlapping between the m-1th probe and the mth probe, and preferably, x2 is any integer between 0 and 100;

[0014] Further preferably, the first probe composition comprises a nucleotide sequence as shown in SEQ ID NO: 13-14 or SEQ ID NO: 15-16; the second probe composition comprises a nucleotide sequence as shown in SEQ ID NO: 17-18 or SEQ ID NO: 19-20.

[0015] 7. Use of a nucleic acid for detecting a marker in preparing a kit for detecting endometrial cancer, wherein the marker is the TBX15 gene or the MAP10 gene.

[0016] 8. The use according to item 7, wherein the target sequence of the TBX15 gene is as shown in any one of SEQ ID NOs: 1-6, or includes the sequence shown in any one of SEQ ID NOs: 1-6; the target sequence of the MAP10 gene is as shown in any one of SEQ ID NOs: 7-12, or includes the sequence shown in any one of SEQ ID NOs: 7-12.

[0017] 9. The use according to item 7 or 8, wherein the nucleic acid is used to target methylated markers of endometrial cancer.

[0018] 10. The use according to any one of items 7 to 9, wherein the nucleic acid is the probe composition according to any one of items 3 to 6.

[0019] 11. The use according to any one of items 7 to 9, wherein the nucleic acid comprises:

[0020] A primer, wherein the primer is a fragment of at least 9 nucleotides in the target sequence of the TBX15 gene or the MAP10 gene, and the fragment contains at least one CpG dinucleotide sequence;

[0021] Preferably, the nucleic acid further comprises:

[0022] A probe that hybridizes with at least a 15-nucleotide fragment in the target sequence of the TBX15 gene or MAP10 gene under moderately stringent or stringent conditions, wherein the fragment contains at least one CpG dinucleotide sequence.

[0023] 12. The use according to any one of items 7 to 11, wherein the nucleic acid further comprises:

[0024] Blockers that preferentially bind to target sequences in their unmethylated state.

[0025] 13. A composition for detecting endometrial cancer, wherein the composition comprises a nucleic acid for detecting methylation of a TBX15 gene or a MAP10 gene.

[0026] 14. The composition according to item 13, wherein the target sequence of the TBX15 gene is as shown in any one of SEQ ID NOs: 1-6, or includes the sequence shown in any one of SEQ ID NOs: 1-6; the target sequence of the MAP10 gene is as shown in any one of SEQ ID NOs: 7-12, or includes the sequence shown in any one of SEQ ID NOs: 7-12.

[0027] 15. The composition according to item 13 or 14, wherein the nucleic acid comprises the probe composition according to any one of items 3 to 6.

[0028] 16. The composition according to item 13 or 14, wherein the nucleic acid comprises:

[0029] A primer, wherein the primer is a fragment of at least 9 nucleotides in the target sequence of the marker, and the fragment contains at least one CpG dinucleotide sequence.

[0030] 17. The composition according to item 16, wherein the nucleic acid further comprises:

[0031] A probe that hybridizes to at least a 15-nucleotide fragment in the target sequence of the marker under moderately stringent or stringent conditions, wherein the fragment contains at least one CpG dinucleotide sequence.

[0032] 18. The composition according to any one of items 3 to 17, further comprising an agent for converting the unmethylated cytosine base at position 5 of the target sequence of the TBX15 gene or the MAP10 gene into uracil.

[0033] 19. The composition according to any one of items 13-18, wherein the nucleic acid further comprises:

[0034] Blockers that preferentially bind to target sequences in their unmethylated state.

[0035] 20. A kit comprising a reagent for detecting the marker described in Item 1 or 2, the probe composition described in any one of Items 3 to 6, or the composition described in any one of Items 13 to 19.

[0036] 21. A chip comprising a reagent for detecting the marker described in Item 1 or 2, the probe composition described in any one of Items 3 to 6, or the composition described in any one of Items 13 to 19.

[0037] Effects of the Invention

[0038] The inventors of the present application used epigenomic and bioinformatics techniques to analyze the whole genome methylation data of endometrial cancer and found two methylation genes related to endometrial cancer. They also determined the target sequences of endometrial cancer methylation genes where methylation abnormalities occur. Furthermore, through the target sequence of either of these two methylation genes, the methylation status of the gene can be sensitively and specifically detected, which can be used to detect cervical exfoliated cell DNA.

[0039] The composition described in the present application is used for screening asymptomatic people in a non-invasive manner, reducing the harm caused by invasive testing. The composition has higher sensitivity and accuracy and can achieve real-time monitoring. DETAILED DESCRIPTION

[0040] The present application is described in detail below. Although specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0041] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the implementation of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

[0042] The present application provides a marker for detecting endometrial cancer, wherein the marker is a TBX15 gene or a MAP10 gene. In some embodiments, the target sequence of the TBX15 gene is as shown in any one of SEQ ID NOs: 1-6, or includes a sequence shown in any one of SEQ ID NOs: 1-6. In some embodiments, the target sequence of the MAP10 gene is as shown in any one of SEQ ID NOs: 7-12, or includes a sequence shown in any one of SEQ ID NOs: 7-12.

[0043] In a specific embodiment, the target sequence of the TBX15 gene is shown in any one of SEQ ID NOs: 1-6.

[0044] The nucleotide sequence of SEQ ID NO:1 is:

[0045]

[0046] The reverse complementary strand sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 2:

[0047]

[0048] In the present application, the above sequences refer to sequences that have not been converted by bisulfite.

[0049] The present application provides a probe composition, wherein the probe composition comprises a probe targeting the methylation of the above-mentioned marker.

[0050] In this application, the methylation refers to the methylation process of the 5th carbon atom on cytosine in CpG dinucleotides. As a relatively stable modification state, it can be inherited to the new offspring DNA during the DNA replication process under the action of DNA methyltransferase. It is an important epigenetic mechanism. When DNA is methylated, the methylation of the gene promoter region can lead to the transcriptional silencing of tumor suppressor genes, so it is closely related to the occurrence of tumors. Abnormal methylation includes high methylation of tumor suppressor genes and DNA repair genes, low methylation of repetitive sequence DNA, and loss of imprinting of certain genes, which are related to the occurrence of various tumors.

[0051] The methylation described herein may be a methylation level, a methylation degree or a methylation state. When analyzing the methylation of such a target sequence, those skilled in the art may use a quantitative measurement method to determine the methylation.

[0052] In the present application, the probe is a single-stranded or double-stranded DNA with a length ranging from tens to hundreds or even thousands of base pairs. It can utilize the molecular denaturation, renaturation and high accuracy of base complementary pairing to hydrogen bond (hybridize) with the complementary non-labeled single-stranded DNA or RNA in the sample to be tested to form a double-stranded complex (hybrid). After washing away the unpaired bound probe, the hybridization reaction result can be detected by a detection system such as radioautography or enzyme-linked reaction. In the present application, the area complementary to or hybridized with the probe is a specific target area, and multiple probes are combined into a probe composition.

[0053] In one embodiment, the probe composition comprises a first hypermethylated probe composition and a second hypomethylated probe composition, wherein the first probe composition is used to hybridize with a region of CG hypermethylation converted by bisulfite, and the second probe composition is used to hybridize with a region of CG hypomethylation converted by bisulfite.

[0054] The CG high methylation region refers to the region where the target sequence is more methylated. Since the methylation status of each person is different, the extreme CG high methylation region refers to the region where all CGs of the target sequence are methylated.

[0055] A CG hypomethylated region refers to a region in which the target sequence is less methylated. In the present application, an extremely CG hypomethylated region refers to a region in which all CGs in the target sequence are not methylated.

[0056] The CG hypermethylated region after bisulfite conversion refers to the conversion of base C to base T after the target sequence is converted by bisulfite, but if it is a base CG, since the 5-methylcytosine residue (5mC) is resistant to it, the C in CG will not be converted, so the base C remains unchanged;

[0057] The CG hypomethylated region after bisulfite conversion means that after bisulfite conversion, since all or most of the bases CG on the target sequence are not methylated, all or most of the bases C are converted to base T.

[0058] Since the methylation status of each person is different, the sequence obtained after bisulfite conversion is also different. Here is an extreme case of this marker, that is, all CGs in this segment are in a high methylation state, and the high methylation state sequence of its complementary chain is shown:

[0059] An extreme case of the hypermethylated state of SEQ ID NO: 1 is shown in SEQ ID NO: 3:

[0060]

[0061] The sequence of the hypermethylated state of the reverse complementary strand of SEQ ID NO: 1 is shown in SEQ ID NO: 4:

[0062]

[0063] Similarly, since the methylation status of each person is different, an extreme case is shown here, that is, all CGs are in a low methylation state, and the low methylation state sequence of its reverse complementary chain is also shown.

[0064] An extreme case of the low methylation state of SEQ ID NO: 1 is shown in SEQ ID NO: 5:

[0065]

[0066] The sequence of the reverse complementary strand of SEQ ID NO:1 in a low methylation state is shown in SEQ ID NO:6:

[0067]

[0068] Those skilled in the art can understand that deleting one or more nucleotides, adding one or more nucleotides, or replacing one or more nucleotides on the basis of the nucleotide sequence shown in SEQ ID NO: 1-6 but having 90%, 91%, 92%, 93%, 94%, 95% or 96% or 97% or 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO: 1-6 can also be used as the methylation marker of the present application and should also be understood to be covered within the scope of protection of the present application.

[0069] In a specific embodiment, the target sequence of the MAP10 gene is shown in any one of SEQ ID NOs: 7-12.

[0070] The nucleotide sequence of SEQ ID NO:7 is:

[0071]

[0072] The reverse complementary strand sequence of SEQ ID NO:7 is shown in SEQ ID NO:8:

[0073]

[0074] An extreme case of the hypermethylated state of SEQ ID NO:7 is shown in SEQ ID NO:9:

[0075]

[0076] The sequence of the hypermethylated state of the reverse complementary strand of SEQ ID NO:7 is shown in SEQ ID NO:10:

[0077]

[0078] An extreme case of the low methylation state of SEQ ID NO:7 is shown in SEQ ID NO:11:

[0079]

[0080] The sequence of the reverse complementary strand of SEQ ID NO:7 in a low methylation state is shown in SEQ ID NO:12:

[0081]

[0082] Those skilled in the art can understand that deleting one or more nucleotides, adding one or more nucleotides, or replacing one or more nucleotides on the basis of the nucleotide sequence shown in SEQ ID NO:7-12 but having 90%, 91%, 92%, 93%, 94%, 95% or 96% or 97% or 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO:7-12 can also be used as the methylation marker of the present application and should also be understood to be covered within the scope of protection of the present application.

[0083] In some embodiments, the first probe composition includes n probes that hybridize to each nucleotide of the sense strand and the antisense strand of the bisulfite-converted CG hypermethylated region.

[0084] The second probe composition includes m probes that hybridize to each nucleotide of the sense strand and / or antisense strand of the bisulfite-converted CG hypomethylated region.

[0085] The present application does not impose any limitation on the number of probes in the first probe composition and the second probe composition, and those skilled in the art may select the number as needed. For example, m and n may be any integer between 1 and 10, and m and n may be the same or different.

[0086] For example, m and n can be any integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, m=n=2.

[0087] In one embodiment, there are x1 nucleotide overlaps between the n-1th probe and the nth probe, preferably, x1 is any integer between 0 and 100;

[0088] Preferably, there are x2 nucleotide overlaps between the m-1th probe and the mth probe, and preferably, x2 is any integer between 0-100.

[0089] Among them, x1 and x2 can be the same or different. When x1 is 0, it indicates that the tail of the n-1th probe is connected to the head of the nth probe. Similarly, when x2 is 0, it indicates that the tail of the m-1th probe is connected to the head of the mth probe.

[0090] The present application hybridizes the probe composition with the target sequence converted by bisulfite, wherein the highly methylated first probe composition hybridizes with the CG highly methylated region, and the low methylated second probe composition hybridizes with the CG low methylated region, thereby being able to efficiently and accurately detect the methylation level of the target sequence, and further being able to be used for endometrial cancer screening.

[0091] In one embodiment, the hypermethylated first probe composition includes one or two of SEQ ID NOs: 13-16.

[0092] The first probe composition for hybridizing with the methylation sequence of the TBX15 gene includes the nucleotide sequence shown in SEQ ID NO: 13-14.

[0093] The nucleotide sequence shown in SEQ ID NO:13 is:

[0094]

[0095] The nucleotide sequence shown in SEQ ID NO:14 is:

[0096]

[0097] The first probe composition for hybridizing with the MAP10 gene methylation sequence includes the nucleotide sequence shown in SEQ ID NO: 15-16.

[0098] The nucleotide sequence shown in SEQ ID NO:15 is:

[0099]

[0100] The nucleotide sequence shown in SEQ ID NO:16 is:

[0101]

[0102] The hypomethylated second probe composition includes one or two of SEQ ID NOs: 17-20.

[0103] The second probe composition for hybridizing with the methylation sequence of the TBX15 gene includes the nucleotide sequences shown in SEQ ID NOs: 17-18.

[0104] The nucleotide sequence shown in SEQ ID NO:17 is:

[0105]

[0106] The nucleotide sequence shown in SEQ ID NO:18 is:

[0107]

[0108] The second probe composition for hybridizing with the MAP10 gene methylation sequence includes the nucleotide sequences shown in SEQ ID NOs: 19-20.

[0109] The nucleotide sequence shown in SEQ ID NO:19 is:

[0110]

[0111] The nucleotide sequence shown in SEQ ID NO:20 is:

[0112]

[0113] The present application provides the use of a nucleic acid for detecting a marker in the preparation of a kit for detecting endometrial cancer, wherein the marker is a TBX15 gene or a MAP10 gene. In some embodiments, the nucleotide sequence of the marker is as shown in any one of SEQ ID NO:1-6. In some embodiments, the nucleotide sequence of the marker is as shown in any one of SEQ ID NO:7-12. In some embodiments, the nucleic acid is used to target a methylated marker for endometrial cancer. In some embodiments, the nucleic acid is the probe composition described above.

[0114] In some embodiments, the nucleic acid comprises:

[0115] A primer, wherein the primer is a fragment of at least 9 nucleotides in the target sequence of the TBX15 gene or the MAP10 gene, and the fragment contains at least one CpG dinucleotide sequence;

[0116] Preferably, the nucleic acid further comprises:

[0117] A probe that hybridizes with at least a 15-nucleotide fragment in the target sequence of the TBX15 gene or MAP10 gene under moderately stringent or stringent conditions, wherein the fragment contains at least one CpG dinucleotide sequence.

[0118] That is, in the present application, when the nucleic acid includes primers and probes, if the sample DNA to be tested is converted using bisulfite, the nucleic acid includes a fragment of at least 9 nucleotides in the sequence after the target sequence of the TBX15 gene or MAP10 gene is converted with bisulfite, and the fragment contains at least one CpG dinucleotide sequence.

[0119] In some embodiments, the nucleic acid further comprises:

[0120] Blockers that preferentially bind to target sequences in their unmethylated state.

[0121] The blocker is used to improve the amplification specificity of the PCR amplification primer. The 5' end of the blocker nucleotide sequence has an overlap region of greater than or equal to 5 nucleotides with the 3' end nucleotide sequence of the forward or reverse primer. The blocker and the forward or reverse primer are complementary to the same strand of the 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°C. The nucleotide sequence of the blocker contains at least one CpG dinucleotide sequence and is complementary to the sequence of the target gene target sequence DNA that has not been methylated after bisulfite conversion. Therefore, when the genomic DNA of the biological sample to be detected is a mixture of methylated and unmethylated states, especially when the DNA in the methylated state is far less than the DNA in the unmethylated state, the DNA in the unmethylated state will preferentially bind to the blocker after bisulfite conversion, thereby inhibiting the binding of the DNA template to the PCR primer, so that PCR amplification does not occur, while the DNA in the methylated state does not bind to the blocker, and thus binds to the primer, PCR amplification occurs, and then the fragments obtained by amplification are directly or indirectly detected.

[0122] The present application provides a composition for detecting endometrial cancer, wherein the composition comprises a nucleic acid for detecting methylation of the TBX15 gene or the MAP10 gene. In some embodiments, the nucleotide sequence of the TBX15 gene is as shown in any one of SEQ ID NO:1-6. In some embodiments, the nucleotide sequence of the MAP10 gene is as shown in any one of SEQ ID NO:7-12. In some embodiments, the nucleic acid comprises the probe composition described above.

[0123] In some embodiments, the nucleic acid comprises:

[0124] A primer, wherein the primer is a fragment of at least 9 nucleotides in the target sequence of the TBX15 gene or the MAP10 gene, and the fragment contains at least one CpG dinucleotide sequence. In some embodiments, the nucleic acid further includes:

[0125] A probe that hybridizes with at least a 15-nucleotide fragment in the target sequence of the TBX15 gene or MAP10 gene under moderately stringent or stringent conditions, wherein the fragment contains at least one CpG dinucleotide sequence.

[0126] In some embodiments, the composition further comprises a reagent for converting the unmethylated cytosine base at position 5 of the target sequence of the TBX15 gene or the MAP10 gene into uracil.

[0127] In the present application, the 5-position unmethylated cytosine base refers to the cytosine that has not received a methyl group on the fifth carbon, that is, the cytosine has not undergone methylation modification.

[0128] In the present application, the reagent may be, for example, bisulfite or the like.

[0129] In some embodiments, the nucleic acid further comprises:

[0130] Blockers that preferentially bind to target sequences in their unmethylated state.

[0131] The present application provides a kit comprising a reagent for detecting the marker described above, the probe composition described above, or the composition described above. In some embodiments, the kit further comprises a container for accommodating a biological sample of a subject. In some embodiments, the kit further comprises instructions for using and interpreting the test results.

[0132] In the present application, the biological sample may be, for example, cervical exfoliated cells.

[0133] The present application does not impose any limitation on the method for detecting the methylation level of the target sequence using the above-mentioned kit, and those skilled in the art can select it as needed. For example, the present application provides a method for detecting the methylation level of the marker target sequence using the above-mentioned kit, which comprises the following steps:

[0134] Collect samples from subjects;

[0135] Extracting and purifying DNA from the sample;

[0136] Constructing a DNA library for sequencing from the purified DNA sample;

[0137] The constructed DNA library is transformed with bisulfite;

[0138] Pre-PCR amplification of the bisulfite-converted DNA library;

[0139] Using the probe composition to perform hybridization capture on the sample amplified by the pre-PCR;

[0140] Amplify the products captured by hybridization using PCR;

[0141] Perform high-throughput next-generation sequencing on the products amplified by PCR and captured by hybridization;

[0142] Analyze the sequencing data to determine the methylation level of the sample;

[0143] The threshold of each marker is calculated based on the methylation status of existing samples, and the disease condition of the patient is judged based on the methylation level of a marker in the sample. If the methylation level of a marker in the sample exceeds the threshold, it is a cancer sample, and if it is lower than the threshold, it is a healthy person sample.

[0144] For example, the present application provides a method for detecting the methylation level of a target sequence of a marker using the above-mentioned kit, which comprises the following steps:

[0145] (1) Collect cervical exfoliated cell samples from subjects;

[0146] (2) extracting and purifying genomic DNA from the sample;

[0147] (3) treating the genomic DNA obtained in step (2) with a reagent to convert the unmethylated cytosine base at position 5 into uracil or other bases, that is, the unmethylated cytosine base at position 5 of the target sequence of the marker is converted into uracil or other bases, and the converted base is different from the unmethylated cytosine base at position 5 in terms of hybridization performance and is detectable;

[0148] (4) contacting the DNA treated in step (3) with a DNA polymerase and a primer of the target sequence of the marker, so that the target sequence of the treated marker is amplified to produce an amplification product or is not amplified; if the target sequence of the treated marker undergoes a DNA polymerization reaction, an amplification product will be produced; if the target sequence of the treated marker does not undergo a DNA polymerization reaction, it will not be amplified;

[0149] (5) detecting the amplified product with a probe;

[0150] (6) Based on the presence or absence of the amplification product, determining the methylation status of at least one CpG dinucleotide of the target sequence of the marker, thereby determining the methylation level of the target sequence of the marker.

[0151] The present application provides a chip, which comprises a reagent for detecting the marker described above or the probe composition described above or the composition described above.

[0152] The chip is also called a gene chip. Its sequencing principle is a hybridization sequencing method, that is, a method of determining the nucleic acid sequence by hybridization with a group of nucleic acid probes of known sequences. Probes of target nucleotides with known sequences are fixed on the surface of a substrate. When the nucleic acid sequence with a fluorescent marker in the solution produces a complementary match with the nucleic acid probe at the corresponding position on the gene chip, a group of probe sequences with completely complementary sequences are obtained by determining the probe position with the strongest fluorescence intensity.

[0153] The chip is prepared mainly by using a glass sheet or a silicon sheet as a carrier, and using in-situ synthesis and micro-matrix methods to arrange oligonucleotide fragments or cDNA as probes in sequence on the carrier.

[0154] The chip described in the present application is based on signal detection of DNA sequence hybridization after bisulfite treatment. The bisulfite treatment converts non-methylated cytosine into uracil, while the methylated cytosine remains unchanged. Uracil is then converted into thymine, and finally chip hybridization is performed. Finally, the type of added base is judged according to the fluorescent color, thereby determining whether the site is methylated.

[0155] The present application provides a method for endometrial cancer screening, comprising:

[0156] Detecting the methylation level of the markers, and

[0157] The risk of the subject suffering from endometrial cancer is judged based on the methylation level, and the marker is the TBX15 gene or the MAP10 gene.

[0158] Example

[0159] The present application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % means wt%, i.e., weight percentage. The reagents or instruments used without indicating the manufacturer are all conventional reagent products that can be obtained commercially.

[0160] Example 1: Screening markers

[0161] 1) Sample collection: A total of 90 cervical exfoliated cell samples from endometrial cancer patients were collected from cooperative hospitals, and 100 cervical exfoliated cell samples from healthy people were collected. The following experiment was conducted (see 1.1 for details).

[0162] 2) Candidate marker analysis: DSS software was used to perform differential analysis of the methylation sites obtained from sequencing data between healthy people and endometrial cancer patients. Filtering criteria: P < 0.05, the difference between the means of the two groups was greater than 0.1. 56 differentially methylated regions were obtained.

[0163] 3) Marker verification: Probe capture was designed for the above 56 differentially methylated regions, and verified using the Boercheng sample data (number of endometrial cancer samples = 100 and number of healthy human samples = 70), and finally 2 markers that can distinguish endometrial cancer from healthy people were obtained. Their sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 7, respectively.

[0164] According to the target sequence region obtained, a probe composition (panel) is customized. The probe composition includes a first probe composition with high methylation and a second probe composition with low methylation, wherein the first probe composition for SEQ ID NO:1 includes two probes, the nucleotide sequences of which are shown in SEQ ID NO:13-14, respectively, and the second probe composition includes two probes, which are shown in SEQ ID NO:17-18, respectively. The first probe composition for SEQ ID NO:7 includes two probes, the nucleotide sequences of which are shown in SEQ ID NO:15-16, respectively, and the second probe composition includes two probes, which are shown in SEQ ID NO:19-20, respectively.

[0165] Then it was verified in cervical exfoliated cell samples, and Qiagen was used in the experiment. Circulating Nucleic Acid Kit was used to prepare cfDNA, and the catalog number is 55114. IDT's xGen Methyl-Seq Lib KIT was used to prepare the library, and the catalog number is 10009824. The experimental detection method is as follows:

[0166] 1.1. DNA extraction and purification

[0167] 1.1.1. Sample preparation:

[0168] 1.1.1.1 Add 360 μl Buffer ATL and 48 μl Proteinase K to the sample, vortex to mix, and incubate in a constant temperature oscillator at 56°C and 800 rpm for 45 min until completely dissolved.

[0169] 1.1.1.2 Add 400 μl Buffer AL. Vortex to mix thoroughly. Incubate in a constant temperature shaker at 56°C and 800 rpm for 10 min.

[0170] 1.1.1.3 Add 400 μl of ethanol (96-100%) and vortex to mix thoroughly.

[0171] 1.1.1.4 Add the mixture to the DNeasy Mini spin column and place in a 2 ml collection tube. Centrifuge at ≥6000xg (8000 rpm) for 1 min. Discard the collection tube.

[0172] 1.1.1.5 Place the centrifuge column in a new 2 ml collection tube and add 500 μl Buffer AW1. Centrifuge at ≥6000 x g (8000 rpm / min) for 1 min. Discard the collection tube.

[0173] 1.1.1.6 Place the centrifuge column in a new 2 ml collection tube, add 500 μl Buffer AW2, centrifuge at 20,000 x g (14,000 pm / min) for 1 min. Discard the collection tube.

[0174] 1.1.1.7 Place the centrifugal column in a new 2 ml collection tube, add 250 μl of anhydrous ethanol (96%-100%), centrifuge at 20,000 x g (14,000 pm / min) for 2 min, and discard the collection tube.

[0175] 1.1.1.8 Transfer the centrifuge column to a new 1.5ml or 2ml centrifuge tube and let it stand at room temperature for 2 minutes.

[0176] 1.1.1.9 Add 100 μl Buffer AE to the center of the centrifuge column membrane to elute the DNA. Incubate at room temperature for 1 minute (15-25°C). Centrifuge at ≥6000xg for 1 minute.

[0177] 1.1.1.10 Add 100 μl of Buffer AE to the center of the centrifugal column membrane to elute the DNA. Incubate at room temperature for 1 minute (15-25°C). Centrifuge at ≥6000xg for 1 minute.

[0178] 1.1.1.11 Transfer the DNA to a labeled 2 ml centrifuge tube and take 1 μl for quantification. Store at -80℃.

[0179] 1.2.Bisulfite conversion and purification:

[0180] 1.2.1 Prepare CT conversion reagent:

[0181] 1.2.1.1 Add 700 μl NF water, 300 μl M-Dilution Buffer and 50 μl M-Dissolving Buffer to a tube of CT conversion reagent, mix well at room temperature, and vortex or shake frequently for 10 min.

[0182] 1.2.1.2 After mixing, divide the mixture into 10 reaction volumes.

[0183] 1.2.2 Bisulfite conversion of DNA library:

[0184] Prepare the reaction system according to the table below.

[0185] Table 1

[0186] Components High concentration sample (1ng-2μg) volume Samples from the previous step 40ul CT conversion reagent 110ul Total volume 150ul

[0187] 1.2.3 Adjust the pipette to 100 μl, gently pipette and mix 6 times, then divide into two tubes and place on the PCR instrument.

[0188] 1.2.4 Set the following program to perform the reaction on the PCR instrument: the heated lid temperature is 105℃.

[0189] Table 2

[0190] temperature time 98℃ 10min 64℃ 2.5h 4℃ ∞

[0191] 1.2.5 Take a new 1.5ml centrifuge tube and add 600μl of M-Binding Buffer.

[0192] 1.2.6 After PCR, transfer the two tubes of the same sample to the corresponding 1.5ml centrifuge tubes mentioned above by brief centrifugation and mix well.

[0193] 1.2.7 Add the mixed sample to Zymo-Spin TM Invert the IC Column to mix, and centrifuge at 10,000 x g for 30 s.

[0194] 1.2.8 Add 100 μl of M-Wash Buffer to the column and centrifuge at 10,000 x g for 30 s.

[0195] 1.2.9 Add 200 μl of M-Desulphonation Buffer to the column, incubate at room temperature for 15-20 min, and centrifuge at 10,000 x g for 30 s.

[0196] 1.2.10 Add 200 μl of M-Wash Buffer to the column and centrifuge at 10,000 x g for 30 s.

[0197] 1.2.11 Repeat the previous step once.

[0198] 1.2.12 Place the column in a new collection tube and centrifuge again at 10,000 x g for 30 seconds.

[0199] 1.2.13 Place the recovery column in a new 1.5 ml EP tube, add 15 μl LOW EDTA buffer to the center of the column membrane, and centrifuge at 10,000 x g for 30 seconds.

[0200] 1.3 Denaturation:

[0201] 1.3.1 Preheat the PCR instrument to 95°C.

[0202] 1.3.2 Set the following program to perform the reaction on the PCR instrument: the heated lid temperature is 105℃.

[0203] Table 3

[0204] temperature time 95℃ ∞ 95℃ 2min 95℃ ∞

[0205] 1.3.3 After incubation, immediately place the tube on ice for 2 minutes.

[0206] 1.4 Connector connection and purification:

[0207] 1.4.1 Configure the reaction system according to the following table:

[0208] Table 4

[0209] Components volume Low EDTA TE 11.5μl Buffer G1 4μl Reagent G2 4μl Reagent G3 2.5μl Enzyme G4 1μl Enzyme G5 1μl Enzyme G6 1μl DNA 15ul Total volume 40ul

[0210] 1.4.2 Set the following program to perform the reaction on the PCR instrument: the heated lid temperature is 105℃.

[0211] Table 5

[0212] temperature time 37℃ ∞ 37℃ 15min 95℃ 2min 4℃ ∞

[0213] 1.5 Sample extension and purification:

[0214] 1.5.1 Configure the reaction system according to the following table:

[0215] Table 6

[0216] Components volume Reagent Y1 2ul Enzyme Y2 42ul totalVolume 44ul

[0217] 1.5.2 Set the following program to perform the reaction on the PCR instrument: the heated lid temperature is 105℃.

[0218] Table 7

[0219] temperature time 98℃ ∞ 98℃ 1min 62℃ 2min 65℃ 5min 4℃ ∞

[0220] 1.5.3 The liquid turns blue after adding DNA protection buffer. Mix by gently pipetting, then divide into two tubes and place on the PCR instrument.

[0221] 1.5.4 Set the following program and run: Heat lid at 105°C.

[0222] Table 8

[0223]

[0224]

[0225] 1.5.5 Prepare the purification system according to the following table:

[0226] Table 9

[0227] Input Reaction volume Magnetic beads amount volume 200bp (SeqCap Epi) 84ul 101μl(ratio:1.2) 15μl

[0228] 1.5.6 Add the above proportion of magnetic beads to each sample for recovery, shake and mix, and centrifuge instantly.

[0229] 1.5.7 Incubate at room temperature for 5 minutes.

[0230] 1.5.8 Mix by shaking and centrifuge briefly. Place on a magnetic rack for adsorption until the solution becomes clear (~2 minutes). After the solution becomes clear, aspirate the supernatant.

[0231] 1.5.9 Add 200 μl of 80% ethanol to wash the beads for 30 s, discard the supernatant and carefully remove all the remaining ethanol from the inner wall of the dropper.

[0232] 1.5.10 Repeat the above steps.

[0233] 1.5.11 Add 15 μl low EDTA TE buffer to elute and shake to mix.

[0234] 1.5.12 Incubate at room temperature for 2 minutes.

[0235] 1.5.13 Place on a magnetic rack and adsorb until the solution becomes clear (~2 min). After the solution becomes clear, aspirate the supernatant.

[0236] 1.5.14 Transfer the entire eluate into a new 0.2 mL PCR tube, making sure that the eluate does not contain the magnetic beads.

[0237] 1.6 Connector ligation and purification:

[0238] 1.6.1 Prepare the library reaction system according to the following table:

[0239] Table 10

[0240] Components volume Buffer B1 3ul Reagent B2 10μl Enzyme B3 2ul TotalVolume 15ul

[0241] 1.6.2 Set up the following programs and run them:

[0242] Table 11

[0243]

[0244]

[0245] 1.6.3 Prepare the purification system according to the following table:

[0246] Table 12

[0247] Input Reaction volume Magnetic beads amount volume 200bp (SeqCap Epi) 30ul 36μl(ratio:1.2) 20μl

[0248] 1.6.4 Add the above proportion of magnetic beads to each sample for recovery, shake and mix, and centrifuge instantly.

[0249] 1.6.5 Incubate at room temperature for 5 minutes.

[0250] 1.6.6 Mix by shaking and centrifuge briefly. Place on a magnetic rack for adsorption until the solution becomes clear (~2 min). After the solution becomes clear, aspirate the supernatant.

[0251] 1.6.7 Add 200 μl of 80% ethanol to wash the beads for 30 s, discard the supernatant and carefully remove any remaining ethanol from the inner wall of the dropper.

[0252] 1.6.8 Repeat the above cleaning steps.

[0253] 1.6.9 Add 15 μl low EDTA TE buffer to elute and shake to mix.

[0254] 1.6.10 Incubate at room temperature for 2 min.

[0255] 1.6.11 Place on a magnetic rack and adsorb until the solution becomes clear (~2 min). After the solution becomes clear, aspirate the supernatant.

[0256] 1.6.12 Transfer the entire eluate into a new 0.2 mL PCR tube, making sure that the eluate does not contain the magnetic beads.

[0257] 1.7 Library amplification and purification:

[0258] 1.7.1 Prepare the library reaction system according to the following table:

[0259] Table 13

[0260] Components volume The above reaction DNA 20ul KAPA HiFi HotStart Uracil+ReadyMix(2x) 25ul index(U001-U024) 5μl Total volume 50ul

[0261] 1.7.2. Set the following program and run: Heated lid 105℃:

[0262] Table 14

[0263]

[0264]

[0265] 1.7.3 The recommended number of cycles is as follows:

[0266] Table 15

[0267] Input Recommended number of cycles 20ng cfDNA 10-11 100ng gDNA 9-10 20ng gDNA 11-12

[0268] 1.7.4 Prepare the purification system according to the following table:

[0269] Table 16

[0270] Input Reaction volume Magnetic beads amount volume 200bp (SeqCap Epi) 50ul 60μl(ratio:1.2) 22μl

[0271] 1.7.5 Transfer the PCR product into a 1.5 ml centrifuge tube.

[0272] 1.7.6 Add the above proportion of magnetic beads to each sample for recovery, shake and mix, and centrifuge instantly.

[0273] 1.7.7 Incubate at room temperature for 5 min.

[0274] 1.7.8 Mix by shaking and centrifuge briefly. Place on a magnetic rack for adsorption until the solution becomes clear (~2 minutes). After the solution becomes clear, aspirate the supernatant.

[0275] 1.7.9 Add 500 μl of 80% ethanol to wash the beads for 30 s, discard the supernatant and carefully remove any remaining ethanol from the inner wall of the dropper.

[0276] 1.7.10 Repeat the above steps.

[0277] 1.7.11 Place on the magnetic stand for 5-10 minutes until the beads are dry (avoid over-drying, which may result in reduced DNA recovery).

[0278] 1.7.12 Add 15 μl low EDTA TE buffer to elute, then shake to mix.

[0279] 1.7.13 Incubate at room temperature for 2 min.

[0280] 1.7.14 Place on a magnetic rack and adsorb until the solution becomes clear (~2 min). After the solution becomes clear, aspirate the supernatant.

[0281] 1.7.15 Transfer the entire eluate into a new 0.2 mL PCR tube, making sure the eluate does not contain the magnetic beads.

[0282] 1.7.16 Pipette out 1ul for qubit calibration and perform 2100 quality inspection.

[0283] 1.8 Sample and probe hybridization:

[0284] 1.8.1 Mixed samples:

[0285] 1.8.1.1 DNA library dosage refers to the table below. The total dosage can be more than 1500ng, but not more than 4ug;

[0286] Table 17

[0287] Number of mixed samples The amount of each library Total library volume per reaction 1 500ng 500ng 2 500ng 1000ng 3 500ng 1500ng 4 375ng 1500ng 8 187.5ng 1500ng

[0288] 1.8.1.2 Calculate the amount of different samples and mix them evenly in a centrifuge tube.

[0289] 1.8.1.3 Add the following pre-hybridization reagents to the mixed sample and mix well, trying not to create bubbles.

[0290] Table 18

[0291] Components volume Twist probe panel 4ul Universal sealant 8ul Blocking agent solution 5μl Methylation Enhancer 2ul

[0292] 1.8.1.4 Dry the mixed pre-hybridization reagents in a vacuum concentrator at room temperature (if heating is required, please use low temperature).

[0293] 1.8.2 Hybridization:

[0294] 1.8.2.1 Incubate Fast Hybridization Mix at 65℃ for 10min or until all precipitates are dissolved. Vortex quickly and add 20ul of freeze-dried sample to resuspend the sample (do not allow the hybridization solution to return to room temperature). Mix by flicking with your fingertips to avoid creating bubbles.

[0295] 1.8.2.2 Rapidly centrifuge to remove bubbles and add 30ul Hybridization Enhancer to the surface of the above reagents.

[0296] 1.8.2.3 Place the PCR tube into the preheated PCR instrument for hybridization.

[0297] 1.8.2.4 Set the following program and run: Heat lid at 85°C.

[0298] Table 19

[0299] temperature time 95℃ ∞ 95℃ 5min 60℃ 15min-4h

[0300] 1.8.3 Combination:

[0301] 1.8.3.1 Shake the pre-equilibrated streptavidin magnetic beads until completely mixed, and add 100 μl of magnetic beads to a 1.5 ml centrifuge tube.

[0302] 1.8.3.2 Add 200ul of binding buffer and mix well by pipetting.

[0303] 1.8.3.3 Place the centrifuge tube on the magnetic rack for 1 minute or until the solution is clear, discard the supernatant and remove the centrifuge tube.

[0304] 1.8.3.4 Repeat the above washing steps 2 times, for a total of 3 times.

[0305] 1.8.3.5 After the last wash, add 200ul of binding buffer and resuspend by vortexing to mix thoroughly.

[0306] 1.8.3.6 After hybridization, open the lid of the PCR instrument and quickly transfer all the hybridization solution to the balanced magnetic beads.

[0307] 1.8.3.7 Mix the magnetic beads with hybridization solution thoroughly on a shaker, rocker or rotator at room temperature for 30 minutes.

[0308] 1.8.3.8 Remove the centrifuge tube from the mixer, centrifuge quickly and place it on the magnetic rack for 1 minute, remove the supernatant and remove the tube.

[0309] 1.8.3.9 Add 200ul preheated washing solution 1 and mix well.

[0310] 1.8.3.10 Incubate at 63℃ or 65℃ for 5 min.

[0311] 1.8.3.11 Place the centrifuge tube on the magnetic rack for 1 minute, remove the supernatant and take out the tube.

[0312] 1.8.3.12 Repeat the above steps, add 200ul of preheated Wash Buffer 1 again and mix well.

[0313] 1.8.3.13 Incubate at 63°C or 65°C for 5 min.

[0314] 1.8.3.14 Transfer the liquid to a new tube; place it on the magnetic rack for 1 min, remove the supernatant and take out the tube.

[0315] 1.8.3.15 Add 200ul preheated wash buffer 2 and mix well with a pipette tip.

[0316] 1.8.3.16 Incubate at 48°C for 5 min.

[0317] 1.8.3.17 Place on the magnetic rack for 1 min, remove the supernatant and take out the tube.

[0318] 1.8.3.18 Repeat (steps 3.15-3.17) and wash twice, for a total of three times.

[0319] 1.8.3.19 For the last time, use a 10ul pipette tip to suck up the wash solution.

[0320] 1.8.3.20 Add 45ul of water, mix well, and incubate the solution on ice.

[0321] 1.8.4 Post-capture PCR amplification, purification and quality inspection:

[0322] 1.8.4.1 Set the following program and run: Heat lid at 105°C.

[0323] Table 20

[0324]

[0325] 1.8.4.2 Mix the magnetic bead mixture in 1.3 and pipette 22.5ul into a 0.2ml PCR tube.

[0326] 1.8.4.3 Add 2.5ul amplification primers and 25ul KAPA HiFi HotStartReadyMix to a 0.2ml PCR tube, for a total of 50ul reaction system.

[0327] 1.8.4.4 Mix gently with a pipette tip, centrifuge quickly, place in PCR instrument and start amplification.

[0328] 1.8.4.5 Vortex to thoroughly mix the pre-equilibrated DNA purification magnetic beads.

[0329] 1.8.4.6 Add 90ul (1.8*) DNA purification magnetic beads to the amplified PCR product and vortex to mix thoroughly.

[0330] 1.8.4.7 Incubate at room temperature for 5 min.

[0331] 1.8.4.8 Place the centrifuge tube on the magnetic rack for 1 minute and remove the supernatant after the solution becomes clear.

[0332] 1.8.4.9 Without removing the centrifuge tube from the magnetic rack, directly add 200ul of freshly prepared 80% ethanol, incubate for 1 minute, and discard the supernatant; repeat the 80% ethanol wash once (2 times in total) and keep the centrifuge tube on the magnetic rack.

[0333] 1.8.4.10 Carefully remove the remaining ethanol with a 10ul pipette tip and leave at room temperature for 5-10 minutes or until the magnetic beads are dry. Be careful not to overdry the magnetic beads.

[0334] 1.8.4.11 Remove the tube from the magnetic rack and add 32ul of water, mix thoroughly with a pipette tip, and incubate at room temperature for 2min

[0335] 1.8.4.12 Place the centrifuge tube on the magnetic rack for 3 minutes or until the solution becomes clear.

[0336] 1.8.4.13 Transfer 30ul of supernatant to a clean 0.2ml centrifuge tube.

[0337] 1.8.4.14 Take 1 μl of library and use Qubit to quantify, and record the library concentration.

[0338] 1.8.4.15 Take 1 μl of sample and use Agilent2100 to measure the library fragment length.

[0339] 1.8.4.16 Sequencing was performed using the Illumina high-throughput sequencing platform.

[0340] 1.9. Methylation bioinformatics analysis process.

[0341] The process is as follows: Use fastp quality control software to check the sequencing quality, remove low-quality reads, then use Bismark alignment software to align the clean data after quality control to the reference genome, and use Bismar_methylation_extractor software to extract the corresponding methylation sites. Finally, calculate the methylation level of the target region. If the value exceeds the threshold, it is judged as cancer, and if it is lower than the threshold, it is judged as normal.

[0342] 1.10. Based on the training set of 90 endometrial cancer samples and 100 normal human samples, the methylation library construction method described in Example 1 was used to screen out biomarkers related to endometrial cancer by using the differences in methylation levels in different groups (endometrial cancer and normal), and the site data was verified in normal and endometrial cancer samples by independent data sets, and the two DNA fragments that most significantly distinguished normal and cancer samples were screened out. The methylation biomarkers (hereinafter referred to as sites or markers) and the discrimination thresholds are shown in Table 21 below;

[0343] The calculation method for the methylation level threshold is: draw the ROC curve according to the data set (including the type and methylation level of each sample), and the confusion matrix corresponding to the optimal threshold point on the ROC curve will be the basis for us to calculate indicators such as sensitivity, specificity, and accuracy. Usually we will make a selection through the Youden index. The Youden index, also known as the correct index, refers to the sum of sensitivity and specificity minus 1: Youden index = Sensitivity + Specificity – 1. The Youden index ranges from 0 to 1, representing the total ability of the classification model to detect true patients and non-patients. The larger the Youden index, the better the performance of the classification model:

[0344] Table 21: Specific performance data of methylation markers

[0345] SEQ ID Threshold Specificity Sensitivity AUC SEQ ID NO.1 0.201 0.943 0.890 0.912 SEQ ID NO.2 0.153 0.914 0.850 0.885

[0346] Example 2

[0347] 6 human samples (S1-3 are healthy samples, S4-6 are endometrial cancer patient samples), the methylation marker detection method of the present application is adopted, and cervical exfoliated cell samples are collected according to the method of Example 1; a library is built, and sequenced by the Illumina platform; the sequencing data is subjected to the above-mentioned bioinformatics analysis process to obtain the methylation level of each marker, and according to the threshold value of each marker, the disease condition of the patient is predicted. If it exceeds the threshold value, it is an endometrial cancer sample, and if it is below the threshold value, it is a healthy person sample. The specific results are as follows:

[0348] The judgment results are as follows: 0 represents normal classification, that is, healthy; 1 represents abnormal classification, that is, tumor.

[0349] Table 22

[0350]

[0351] As can be seen from Table 22, the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 7 can specifically detect endometrial cancer patient samples.

[0352] In summary, the inventors of the present application have obtained a methylation gene associated with endometrial cancer and determined the target sequence of the endometrial cancer methylation gene for abnormal methylation. Moreover, through the target sequence of the methylation gene, the methylation state of the gene can be sensitively and specifically detected, thereby being used for the detection of cervical exfoliated cell DNA. The composition described in the present application can achieve real-time monitoring with higher sensitivity and accuracy.

[0353] The above is only the preferred embodiment of the present application, and does not limit the present application in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. A marker for detecting endometrial cancer, wherein: The marker is the TBX15 gene or the MAP10 gene.

2. The marker according to claim 1, wherein The target sequence of the TBX15 gene is shown in any one of SEQ ID NOs: 1-6, or includes the sequence shown in any one of SEQ ID NOs: 1-6; the target sequence of the MAP10 gene is shown in any one of SEQ ID NOs: 7-12, or includes the sequence shown in any one of SEQ ID NOs: 7-12.

3. A probe composition, wherein: The probe composition comprises a probe targeting the methylation of the marker according to claim 1 or 2.

4. The probe composition according to claim 3, wherein The probe composition comprises a first hypermethylated probe composition and a second hypomethylated probe composition, wherein the first probe composition is used for hybridizing with a CG hypermethylated region converted by bisulfite, and the second probe composition is used for hybridizing with a CG hypomethylated region converted by bisulfite.

5. The probe composition according to claim 4, wherein The first probe composition comprises n probes, and the n probes hybridize with each nucleotide of the sense chain and / or antisense chain of the CG high methylation region converted by bisulfite. Preferably, the second probe composition comprises m probes, and the m probes hybridize with each nucleotide of the sense chain and / or antisense chain of the CG low methylation region converted by bisulfite. Further preferably, n and m are any integers between 1 and 10.

6. The probe composition according to claim 5, wherein There are x1 nucleotide overlaps between the n-1th probe and the nth probe, preferably, x1 is any integer between 0 and 100; Preferably, there are x2 nucleotides overlapping between the m-1th probe and the mth probe, and preferably, x2 is any integer between 0 and 100; Further preferably, the first probe composition comprises a nucleotide sequence as shown in SEQ ID NO: 13-14 or SEQ ID NO: 15-16; the second probe composition comprises a nucleotide sequence as shown in SEQ ID NO: 17-18 or SEQ ID NO: 19-20.

7. Use of a nucleic acid for detecting a marker in the preparation of a kit for detecting endometrial cancer, wherein: The marker is the TBX15 gene or the MAP10 gene.

8. The use according to claim 7, wherein The target sequence of the TBX15 gene is shown in any one of SEQ ID NOs: 1-6, or includes the sequence shown in any one of SEQ ID NOs: 1-6; the target sequence of the MAP10 gene is shown in any one of SEQ ID NOs: 7-12, or includes the sequence shown in any one of SEQ ID NOs: 7-12.

9. The use according to claim 7 or 8, wherein The nucleic acid is used to target methylated markers of endometrial cancer.

10. The use according to any one of claims 7 to 9, wherein The nucleic acid is the probe composition according to any one of claims 3 to 6.

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