Methylation marker and probe composition for detecting cervical cancer and application of methylation marker and probe composition

Abstract

The invention discloses a methylation marker for detecting cervical cancer, a probe composition and application of the methylation marker and the probe composition. The methylation marker is a KHDRBS2 gene. The methylation marker can sensitively and specifically detect the methylation state of the gene, so that the methylation marker can be used for detecting DNA of cervical exfoliated cells, and the composition has higher sensitivity and accuracy and can realize real-time monitoring.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a methylation marker, a probe composition for detecting cervical cancer, and their applications. Background Art

[0002] Cervical cancer is one of the common gynecological malignancies. In the prior art, the diagnosis and treatment methods for cervical cancer include cervical / vaginal cytological smear examination and HPV detection, but each has certain deficiencies. For cervical / vaginal cytological smear examination, mainly thin-prep cytology test (TCT) is used. Cervical cell samples are collected by using a special sampler for TCT, and the sampler is placed into a vial containing cell preservation solution for rinsing. Subsequently, a fully automatic cell detector is used to disperse and filter the sample to reduce the traces of blood, mucus, and inflammatory tissues. Finally, the sample is subjected to microscopic detection and diagnosis. However, TCT detection is relatively dependent on the experience of cytopathologists. Affected by the patient's personal physiological conditions and sampling positions, it will cause certain difficulties in the interpretation of results. In addition, false negatives may occur in some special types of cervical cancer. For HPV detection, according to the risk of causing cervical cancer, HPV can be divided into two major categories: high-risk types and low-risk types. The high-risk types include types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 of HPV, and the low-risk types include types 6, 11, 40, 42, 43, 44, 53, 54, 61, 72, 73, and 81 of HPV. The vast majority of cervical cancers are caused by HPV infection, among which HPV type 16 accounts for about 50%, and HPV type 18 accounts for about 20%. However, not all HPV type 16 or 18 infected patients will progress to cancer. The specificity of HPV detection is insufficient, and continuous positive results will cause panic among patients, leading to a sharp increase in the number of colposcopy examinations in hospitals and even over-medical treatment.

[0003] In addition, the occurrence of cervical cancer can be effectively controlled by the examination and treatment of precancerous lesions. During the process of cervical intraepithelial neoplasia (CIN) developing into cervical cancer, there is abnormal expression of specific gene methylation. Detecting the degree of gene methylation can be used as an important indicator for cervical cancer transformation. When detecting transformational lesions (partial CIN2 and CIN3) through gene methylation detection, the risk of cancer progression can be known, prompting doctors whether to immediately intervene in treatment or maintain observation and follow-up. Therefore, DNA methylation detection can contribute to the early detection of cancer, risk stratification, and the formulation of treatment plans. Summary of the Invention

[0004] Based on this, the present application provides a marker, a probe composition for detecting cervical cancer, and their applications, which can be used for the screening of cervical cancer.

[0005] The specific technical solution of the present application is as follows:

[0006] 1. A methylation marker for detecting cervical cancer, wherein the methylation marker is the KHDRBS2 gene.

[0007] 2. The methylation marker according to item 1, wherein the target sequence of the methylation marker is shown as any one of SEQ ID NOs: 1-6 or contains a sequence shown as any one of SEQ ID NOs: 1-6.

[0008] 3. A probe composition, wherein the probe composition contains a probe targeting the methylation of the target sequence of the methylation marker described in item 1 or 2.

[0009] 4. The probe composition according to item 3, wherein the probe composition contains a first probe composition for high methylation and a second probe composition for low methylation. The first probe composition is used for hybridizing with the region of CG highly methylated after bisulfite conversion, and the second probe composition is used for hybridizing with the region of CG low methylated after bisulfite conversion;

[0010] Preferably, the first probe composition includes n probes, and the n probes hybridize with each nucleotide of the sense strand and / or antisense strand of the region of CG highly methylated after bisulfite conversion. Preferably, the second probe composition includes m probes, and the m probes hybridize with each nucleotide of the sense strand and / or antisense strand of the region of CG low methylated after bisulfite conversion. Further preferably, both n and m are any integers from 1 to 10;

[0011] Preferably, there are x1 nucleotides overlapping between the (n-1)th probe and the nth probe, and preferably, x1 is any integer from 0 to 100;

[0012] Preferably, there are x2 nucleotides overlapping between the (m-1)th probe and the mth probe, and preferably, x2 is any integer from 0 to 100;

[0013] Further preferably, the first probe composition includes the nucleotide sequences shown in SEQ ID NO: 7-8; the second probe composition includes the nucleotide sequences shown in SEQ ID NO: 9-10.

[0014] 5. Use of a nucleic acid for detecting the target sequence of a methylation marker in the preparation of a kit for detecting cervical cancer, wherein the methylation marker is the KHDRBS2 gene.

[0015] 6. Use according to item 5, wherein the target sequence of the methylation marker is as shown in any one of SEQ ID NOs: 1-6 or contains a sequence as shown in any one of SEQ ID NOs: 1-6;

[0016] Preferably, the nucleic acid is used for targeting the target sequence of the methylation marker after methylation in cervical cancer;

[0017] Preferably, the nucleic acid is the probe composition described in any one of items 3-4.

[0018] 7. Use according to any one of items 5-6, wherein the nucleic acid comprises:

[0019] Primers, the primers being fragments of at least 9 nucleotides in the target sequence of the KHDRBS2 gene, the fragment containing at least one CpG dinucleotide sequence;

[0020] Preferably, the nucleic acid further comprises:

[0021] Probes, the probes hybridizing with at least 15 nucleotide fragments in the target sequence of the KHDRBS2 gene under medium stringency or stringent conditions, the fragment containing at least one CpG dinucleotide sequence;

[0022] Preferably, the nucleic acid further comprises:

[0023] Blockers that preferentially bind to target sequences in the unmethylated state.

[0024] 8. A composition for detecting cervical cancer, wherein the composition comprises a nucleic acid for detecting methylation of the KHDRBS2 gene.

[0025] 9. The composition according to item 8, wherein the target sequence of the KHDRBS2 gene is as shown in any one of SEQ ID NOs: 1-6 or contains a sequence as shown in any one of SEQ ID NOs: 1-6;

[0026] Preferably, the nucleic acid comprises the probe composition described in any one of items 3-4.

[0027] 10. The composition according to item 8 or 9, wherein the nucleic acid comprises:

[0028] Primers, the primers being fragments of at least 9 nucleotides in the target sequence of the methylation marker, the fragment containing at least one CpG dinucleotide sequence;

[0029] Preferably, the nucleic acid further comprises:

[0030] A probe, which hybridizes with at least 15 nucleotide fragments in the target sequence of the methylation marker under medium stringency or stringent conditions, and the fragment contains at least one CpG dinucleotide sequence.

[0031] 11. The composition according to any one of items 8-10, further comprising a reagent for converting the unmethylated cytosine base at the 5th position of the target sequence of the KHDRBS2 gene into uracil;

[0032] Preferably, the nucleic acid further comprises:

[0033] A blocker that preferentially binds to the target sequence in the unmethylated state.

[0034] 12. The composition according to any one of items 8-11, wherein the methylation status of the methylation marker is characterized by the methylation of the target sequence of the methylation marker.

[0035] 13. A kit, which contains a reagent for detecting the methylation marker described in item 1 or 2, the probe composition described in items 3-4, or the composition described in any one of items 8-12.

[0036] 14. A chip, which contains a reagent for detecting the methylation marker described in item 1 or 2, the probe composition described in items 3-4, or the composition described in any one of items 8-12.

[0037] Effects of the Invention

[0038] The methylation marker described in this application has the following technical effects:

[0039] 1. Early detection and diagnosis of cervical cancer: The DNA methylation marker detection technology adopted in this application can detect and diagnose cervical cancer in the early stage. Because DNA methylation markers can change in the early stage of cervical cancer, the accuracy and sensitivity of early screening for cervical cancer can be improved, thereby effectively improving the treatment effect and survival rate.

[0040] 2. Avoiding the defects of traditional cervical cancer screening methods: Traditional cervical cancer screening methods such as cervical / vaginal cytology smear examination and HPV detection have some defects, such as relying on doctor experience, low accuracy, insufficient specificity, etc. Detecting with methylation markers in this application can avoid these defects and also improve the sensitivity and specificity of cervical cancer screening.

[0041] 3. Improved the treatment effect and prognosis of cervical cancer: By using methylation markers for detection in this application, cervical cancer can be detected and diagnosed at an early stage, thus improving the treatment effect and prognosis of cervical cancer. Since early-stage cervical cancer is easily curable, detecting cervical cancer at an early stage can prevent the deterioration and metastasis of the disease, thereby improving the treatment effect and survival rate.

[0042] By using the methylation markers for detection in this application, the accuracy and sensitivity of early screening for cervical cancer can be improved, avoiding the defects of traditional cervical cancer screening methods, and improving the treatment effect and prognosis of cervical cancer. At the same time, by using the methylation markers for detection in this application, it can be used for the auxiliary diagnosis of high-grade cervical lesions or cervical cancer and the management of HPV-positive triage without invasiveness, and has high sensitivity and specificity for advanced transformed CIN2 / 3 or cervical cancer, being more accurate and efficient.

[0043] In addition, the inventors of this application utilized epigenomic and bioinformatics techniques. By analyzing the whole-genome methylation data of cervical cancer, a methylation gene related to cervical cancer was found, and the target sequence of abnormal methylation of the cervical cancer methylation gene was determined. Moreover, through the target sequence of this methylation gene, the methylation status of this gene can be detected sensitively and specifically, and thus it can be used for the detection of DNA in cervical exfoliated cells.

[0044] The composition described in this application has higher sensitivity and specificity (when the AUC is 0.953, the corresponding sensitivity and specificity are 88.31% and 93.97% respectively), and can achieve real-time monitoring. Detailed Description of the Invention

[0045] The present invention will be described in detail below. Although specific embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided so that the present invention can be understood more thoroughly and the scope of the present invention can be fully conveyed to those skilled in the art.

[0046] It should be noted that in the description of the specification and claims, certain terms are used to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. The description of the specification and claims does not use the difference in nouns as a way to distinguish components, but uses the difference in the functions of components as the criterion for distinction. For example, the terms "comprising" or "including" mentioned throughout the specification and claims are open-ended terms, so they should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment for implementing the present invention, but the description is for the purpose of the general principles of the specification and is not used to limit the scope of the present invention. The scope of protection of the present invention shall be determined by the scope defined by the appended claims.

[0047] Unless otherwise specifically defined elsewhere in this specification, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application belongs.

[0048] As used herein, "DNA methylation" of the present application refers to the addition of a methyl group to the 5-position of cytosine (C), which typically (but not necessarily) occurs in the context of CpG (guanine following cytosine) dinucleotides. As used herein, "increased degree of methylation" or "significant degree of methylation" refers to the presence of at least one methylated cytosine nucleotide in a DNA sequence, where the corresponding C in a normal control sample (e.g., a DNA sample extracted from a non-cancerous cell or tissue sample or a DNA sample treated for methylation of DNA residues) is non-methylated. In certain embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Cs may be methylated, where the Cs at these positions in the control DNA sample are non-methylated.

[0049] "Homology", "identity", and "similarity" as used in this application are used interchangeably and refer to sequence similarity between two nucleic acid molecules. Positions within each sequence can be compared to determine "homology", "identity", or "similarity", and the sequences can be aligned for the purpose of comparison. When the equivalent positions in the sequences being compared are occupied by the same base, the molecules are identical at that position; when the equivalent sites are occupied by the same or similar amino acid (e.g., similar in spatial or charged properties) residues, the molecules can be said to be homologous (similar) at that position. The expression of the percentage of homology / similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the sequences being compared. "Unrelated" or "non-homologous" sequences share less than 40% identity, preferably less than 25% identity, with the sequences of this application. When comparing two sequences, the presence of deletions or additional residues (amino acids or nucleic acids) also reduces identity and homology / similarity. In a specific embodiment, for two or more sequences or subsequences, determination is made by using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters or by manual alignment and visual inspection provided, for example, online by the National Center for Biotechnology Information (NCBI). When comparing and aligning for maximum correspondence over a comparison window or specified region, if their sequences have an identity of about 60%, or about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher over the specified region, they can be considered to be substantially or significantly homologous, similar, or identical. This definition also pertains to or can be used to test the complement of a sequence. Thus, to the extent permitted by the context herein, for example, if a nucleotide sequence can be predicted to occur naturally in a DNA duplex or can occur naturally in the form of one or both of the complementary strands, a nucleotide sequence complementary to a specified target sequence or its variant is itself considered to be "similar" to the target sequence, and when referring to "similar" nucleic acid sequences, includes single-stranded sequences, their complementary sequences, double-stranded strand complexes, sequences capable of encoding the same or similar polypeptide products, and any permissible variants of any of the foregoing. Circumstances where similarity must be restricted to the analysis of a single nucleic acid strand sequence can include, for example, the detection and quantification of the expression of a specific RNA sequence or coding sequence in a cell. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions.In embodiments, the identity or similarity can be over a region of at least about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides, or over a region of more than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more than about 100 nucleotides.

[0050] The "sensitivity" of this application represents the proportion of cancers detected from a certain cancer sample, and its calculation formula is: sensitivity = (detected cancers / all cancers), while the "specificity" represents the proportion of normals detected in a certain normal human sample, and its calculation formula is specificity = (detected negatives / total negatives).

[0051] According to the cytology (TCT) test results, cervical precancerous lesions can be classified into: low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL). According to the histological biopsy results, cervical intraepithelial neoplasia includes CIN1, CIN2, and CIN3. CIN 1 is a low-grade lesion, referring to mild atypical cytological changes in the lower 1 / 3 layer of the epithelium. CIN 2 is a high-grade lesion, referring to moderate atypical cytological changes limited to the lower 2 / 3 layer of the epithelium with normal epithelial maturity. CIN 3 is a high-grade lesion, referring to severe atypical cytological changes with the lesion range exceeding the lower 2 / 3 layer of the epithelium and even involving the entire layer.

[0052] According to the International Federation of Gynecology and Obstetrics cervical cancer clinical staging standard (FIGO 2018), the description of stage I cervical cancer is - the tumor is strictly confined to the cervix.

[0053] In this study, the research samples were divided into two training sets: early-stage cervical cancer and normal people. The former included cervical exfoliated cell samples from patients with high-grade cervical precancerous lesions (CIN2, CIN3) and stage I, a total of 98 cases, among which there were 23 CIN2 samples, 39 CIN3 samples, and 36 stage I samples. The latter included samples from healthy people without lesions and low-grade precancerous lesions (CIN1), a total of 85 cases, among which there were 23 healthy person samples and 62 CIN1 samples. Methylation panel detection was performed on the samples, and 165 differentially methylated regions were mined; subsequently, 77 early-stage cervical cancer and 83 healthy person data were used for independent verification, and finally 1 significantly different biomarker for early diagnosis of cervical cancer was screened out.

[0054] When the AUC is 0.953, the corresponding sensitivity and specificity are 88.31% and 93.97% respectively.

[0055] The present application provides a methylation marker for detecting cervical cancer, wherein the methylation marker is the KHDRBS2 gene. In some embodiments, the target sequence of the methylation marker is as shown in any one of SEQ ID NOs: 1-6 or contains a sequence as shown in any one of SEQ ID NOs: 1-6.

[0056] Among them, the sequence of SEQ ID NO: 1 (KHDRBS2 gene) is:

[0057]

[0058] The sequence of SEQ ID NO: 2 (an extreme case of hypermethylation of the sequence of SEQ ID NO: 1) is:

[0059]

[0060] The sequence of SEQ ID NO: 3 (an extreme case of hypomethylation of the sequence of SEQ ID NO: 1) is:

[0061]

[0062] The sequence of SEQ ID NO: 4 (the reverse complementary sequence of SEQ ID NO: 1) is:

[0063]

[0064] The sequence of SEQ ID NO: 5 (an extreme case of hypermethylation of the sequence of SEQ ID NO: 4) is:

[0065]

[0066] The sequence of SEQ ID NO: 6 (an extreme case of hypomethylation of the sequence of SEQ ID NO: 4) is:

[0067]

[0068] In the present application, for sequences SEQ ID NO: 1 and 4, it refers to the sequences without bisulfite conversion.

[0069] In the present application, the methylation refers to the methylation process occurring at the 5th carbon atom of cytosine in CpG dinucleotides. As a relatively stable modification state, under the action of DNA methyltransferase, it can be inherited to the newly generated daughter DNA during DNA replication, and it is an important epigenetic mechanism. When DNA is methylated, the methylation in the gene promoter region can lead to transcriptional silencing of tumor suppressor genes, so it is closely related to the occurrence of tumors. Abnormal methylation includes hypermethylation of tumor suppressor genes and DNA repair genes, hypomethylation of repetitive sequence DNA, and loss of imprinting of certain genes, which is related to the occurrence of various tumors.

[0070] The methylation described in the present application can be methylation level, methylation degree or methylation state. When analyzing the methylation of such target sequences, those skilled in the art can use quantitative determination methods to determine methylation.

[0071] In the present application, the hypermethylation refers to CG hypermethylation. The region of CG hypermethylation refers to the region where the target sequence has more methylation. Since the methylation status of each person is different, the extreme region of CG hypermethylation refers to the region where all CGs of the target sequence are methylated.

[0072] The hypomethylation refers to CG hypomethylation. The region of CG hypomethylation refers to the region where the target sequence has less methylation. In the present application, the extreme region of CG hypomethylation refers to the region where all CGs in the target sequence are not methylated.

[0073] The region of bisulfite-converted CG hypermethylation refers to the region where, after the target sequence is converted by bisulfite, the base C is converted to the base T. However, for the base CG, due to the resistance of 5-methylcytosine residue (5mC), the C in CG will not be converted, so the base C remains unchanged.

[0074] The region of bisulfite-converted CG hypomethylation refers to the region where, after bisulfite conversion, since all or most of the base CGs on the target sequence are not methylated, all or most of the base Cs are converted to the base T.

[0075] Since the methylation status of each person is different, the sequences obtained by bisulfite conversion are also different. Therefore, the present application shows an extreme case of each methylation marker, that is, all CGs in this region are in a hypermethylated state, and shows the hypermethylated state sequence of its complementary strand. Similarly, in the present application, an extreme case of hypomethylation of each methylation marker is also shown. For example, SEQ ID NO: 2 is an extreme case of the hypermethylation of SEQ ID NO: 1. Similarly, SEQ ID NO: 3 is an extreme case of the hypomethylation of SEQ ID NO: 1.

[0076] SEQ ID NO: 4 is the reverse complementary sequence of SEQ ID NO: 1. SEQ ID NO: 5 is an extreme case of hypermethylation of SEQ ID NO: 4, and SEQ ID NO: 6 is an extreme case of hypomethylation of SEQ ID NO: 4.

[0077] Those skilled in the art can also understand that the target sequences of the KHDRBS2 gene are not limited to the specific sequences listed above. The target sequences of the KHDRBS2 gene should cover sequences that contain one, two, or more than three nucleotide mutations compared to any of the sequences shown in SEQ ID NOs: 1-6, but still substantially have the same essential function, and also include sequences that have 95%, 96%, 97%, 98%, or 99% sequence identity compared to any of the sequences shown in SEQ ID NOs: 1-6.

[0078] The present application provides a probe composition, wherein the probe composition comprises a methylated probe targeting the above-mentioned target sequence of the methylation marker.

[0079] In the present application, the probe is a single-stranded or double-stranded DNA with a length of dozens to hundreds or even thousands of base pairs. It can utilize the denaturation, renaturation of molecules and the high precision of base complementary pairing to bind (hybridize) with the complementary non-labeled single-stranded DNA or RNA in the test sample through hydrogen bonds to form a double-stranded complex (hybrid). After washing away the unpaired probes, the results of the hybridization reaction can be detected by detection systems such as autoradiography or enzyme-linked reaction. In the present application, the region complementary to and hybridized with the probe is the specific target region, and multiple probes are combined into a probe composition.

[0080] In one embodiment, the probe composition comprises a first probe composition for hypermethylation and a second probe composition for hypomethylation. The first probe composition is used to hybridize with the CG hypermethylated region after bisulfite conversion, and the second probe composition is used to hybridize with the CG hypomethylated region after bisulfite conversion.

[0081] In some embodiments, the first probe composition includes n probes, and the n probes hybridize with each nucleotide of the sense strand and the antisense strand of the CG hypermethylated region after bisulfite conversion.

[0082] The second probe composition includes m probes, and the m probes hybridize with each nucleotide of the sense strand and / or the antisense strand of the CG hypomethylated region after bisulfite conversion.

[0083] The present invention does not impose any restrictions on the number of probes in the first probe composition and the second probe composition. Those skilled in the art can make selections according to needs. For example, m and n can be any integer from 1 to 10, and m and n can be the same or different.

[0084] For example, m and n can be any integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. Preferably, m = n = 2.

[0085] In one embodiment, there are x1 nucleotide overlaps between the (n - 1)th probe and the nth probe. Preferably, x1 is any integer from 0 to 100;

[0086] Preferably, there are x2 nucleotide overlaps between the (m - 1)th probe and the mth probe. Preferably, x2 is any integer from 0 to 100.

[0087] Among them, x1 and x2 can be the same or different. When x1 is 0, it indicates that the tail of the (n - 1)th probe is connected to the head of the nth probe. Similarly, when x2 is 0, it indicates that the tail of the (m - 1)th probe is connected to the head of the mth probe.

[0088] In this application, the probe composition is hybridized with the target sequence that has been bisulfite - converted. Among them, the first probe composition with high methylation hybridizes with the CG - hypermethylated region, and the second probe composition with low methylation hybridizes with the CG - hypomethylated region, so as to be able to efficiently and accurately detect the methylation level of the target sequence, and further can be used for cervical cancer screening.

[0089] In one embodiment, the first probe composition with high methylation includes the nucleotide sequences shown in SEQ ID NO: 7 - 8. For example, for the KHDRBS2 gene, its first probe composition with high methylation is as shown in SEQ ID NO: 7 and SEQ ID NO: 8.

[0090] Among them, the nucleotide sequence shown in SEQ ID NO: 7 is:

[0091]

[0092] The nucleotide sequence shown in SEQ ID NO: 8 is:

[0093]

[0094] The second probe composition with low methylation includes nucleotide sequences shown in SEQ ID NOs: 9 - 10. For example, for the KHDRBS2 gene, its first probe composition with low methylation is shown in SEQ ID NO: 9 and SEQ ID NO: 10.

[0095] The nucleotide sequence shown in SEQ ID NO: 9 is:

[0096]

[0097] The nucleotide sequence shown in SEQ ID NO: 10 is:

[0098]

[0099] This application provides the use of nucleic acids for detecting methylation markers in the preparation of a kit for detecting cervical cancer, wherein the marker is the KHDRBS2 gene. In some embodiments, the target sequence of the methylation marker is shown in any one of SEQ ID NOs: 1 - 6 or contains a sequence shown in any one of SEQ ID NOs: 1 - 6. In some embodiments, the nucleic acid is used to target the target sequence of the methylation marker after methylation in cervical cancer. In some embodiments, the nucleic acid is the probe composition described above.

[0100] In some embodiments, the nucleic acid includes:

[0101] Primers, which are fragments of at least 9 nucleotides in the target sequence of the KHDRBS2 gene, and the fragment contains at least one CpG dinucleotide sequence;

[0102] Preferably, the nucleic acid further includes:

[0103] Probes, which hybridize with at least 15 - nucleotide fragments in the target sequence of the KHDRBS2 gene under medium stringency or stringent conditions, and the fragment contains at least one CpG dinucleotide sequence.

[0104] That is, in this application, when the nucleic acid includes primers and probes, if bisulfite conversion is used to transform the DNA of the sample to be tested, the nucleic acid includes a fragment of at least 9 nucleotides in the sequence after bisulfite conversion of the target sequence of the KHDRBS2 gene, and the fragment contains at least one CpG dinucleotide sequence.

[0105] In some embodiments, the nucleic acid further includes:

[0106] A blocker that preferentially binds to the target sequence in the non - methylated state.

[0107] The blocker is used to improve the amplification specificity of PCR amplification primers. The 5'-end of the nucleotide sequence of the blocker has an overlapping region of greater than or equal to 5 nucleotides with the 3'-end nucleotide sequence of the forward or reverse primer. The blocker is complementary to the same strand of the target gene target sequence DNA as the forward or reverse primer. 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 unmethylated target gene target sequence DNA after bisulfite conversion. Therefore, when the genomic DNA of the biological sample to be detected is a mixture of methylated and non-methylated states, especially when the DNA in the methylated state is far less than the DNA in the non-methylated state, after the non-methylated DNA is converted by bisulfite, it will preferentially bind to the blocker, thereby inhibiting the binding of the DNA template to the PCR primer, and thus no PCR amplification occurs. However, the DNA in the methylated state does not bind to the blocker, so it binds to the primer and PCR amplification occurs, and then the fragment obtained by amplification is detected directly or indirectly.

[0108] The present application provides a composition for cervical cancer detection, wherein the composition contains a nucleic acid for detecting the methylation of the KHDRBS2 gene. In some embodiments, the target sequence of the KHDRBS2 gene is as shown in any one of SEQ ID NOs: 1-6 or contains the sequence as shown in any one of SEQ ID NOs: 1-6. In some embodiments, the nucleic acid contains the probe composition described above.

[0109] In some embodiments, the nucleic acid includes:

[0110] Primers, the primers are fragments of at least 9 nucleotides in the target sequence of the KHDRBS2 gene, and the fragments contain at least one CpG dinucleotide sequence. In some embodiments, the nucleic acid further includes:

[0111] Probes, the probes hybridize with at least 15 nucleotide fragments in the target sequence of the KHDRBS2 gene under medium stringency or stringent conditions, and the fragments contain at least one CpG dinucleotide sequence.

[0112] In some embodiments, the composition further includes a reagent for converting the 5-position unmethylated cytosine base of the target sequence of the KHDRBS2 gene into uracil.

[0113] In the present application, the 5-position unmethylated cytosine base refers to the fifth carbon of cytosine that does not receive a methyl group, that is, cytosine is not methylated.

[0114] In the present application, the reagent can be, for example, bisulfite.

[0115] In some embodiments, the nucleic acid further comprises:

[0116] A blocker that preferentially binds to a target sequence in a non-methylated state.

[0117] The present application provides a kit, which contains reagents for detecting the above-mentioned marker, the above-mentioned probe composition or the above-mentioned composition. In some embodiments, the kit further comprises a container for accommodating a biological sample of a subject. In some embodiments, the kit further includes instructions for using and interpreting the test results.

[0118] In the present application, the biological sample may be, for example, cervical secretions or exfoliated cervical cells.

[0119] The present application does not impose any restrictions on the method for detecting the methylation level of a target sequence using the above-mentioned kit, and those skilled in the art can make selections according to needs. For example, the present application provides a method for detecting the methylation level of a target sequence of a methylation marker using the above-mentioned kit, which includes the following steps:

[0120] Collect a sample from a subject;

[0121] Extract and purify the DNA in the sample;

[0122] Construct a DNA library for sequencing for the purified DNA sample;

[0123] Bisulfite convert the constructed DNA library;

[0124] Pre-PCR amplify the bisulfite-converted DNA library;

[0125] Use the probe composition to perform hybridization capture on the pre-PCR amplified sample;

[0126] PCR amplify the product after hybridization capture;

[0127] Perform high-throughput next-generation sequencing on the product after hybridization capture and PCR amplification;

[0128] Analyze the sequencing data to determine the methylation level of the sample;

[0129] Calculate the threshold for each marker based on the methylation status of the existing samples, and judge the disease status of the patient based on the methylation level of the marker in the sample. If the methylation level of the marker in the sample exceeds the threshold, it is a cancer sample; if it is lower than the threshold, it is a healthy person sample.

[0130] For another example, the present application provides a method for detecting the methylation level of a target sequence of a methylation marker using the above-mentioned kit, which comprises the following steps:

[0131] (1) Scraping cervical secretions to obtain cervical exfoliated cells;

[0132] (2) Extracting genomic DNA from the cervical exfoliated cells;

[0133] (3) Treating the genomic DNA obtained in step (2) with a reagent to convert the cytosine base unmethylated at the 5th position into uracil or other bases, that is, converting the cytosine base unmethylated at the 5th position of the target sequence of the marker into uracil or other bases. The converted base is different from the cytosine base unmethylated at the 5th position in terms of hybridization performance and is detectable;

[0134] (4) Contacting the genomic DNA treated in step (3) with a DNA polymerase and a primer of the target sequence of the marker, so that the treated target sequence of the marker is amplified to produce an amplification product or not amplified; if the treated target sequence of the marker undergoes a DNA polymerization reaction, an amplification product will be produced; if the treated target sequence of the marker does not undergo a DNA polymerization reaction, it will not be amplified;

[0135] (5) Detecting the amplification product with a probe;

[0136] (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.

[0137] The present application provides a chip, which comprises a reagent for detecting the above-mentioned marker or the above-mentioned probe composition or the above-mentioned composition.

[0138] The chip is also called a gene chip, and its sequencing principle is the hybridization sequencing method, that is, a method for nucleic acid sequence determination by hybridizing with a group of nucleic acid probes with 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 label in the solution produces complementary matching with the nucleic acid probe at the corresponding position on the gene chip, by determining the position of the probe with the strongest fluorescence intensity, a group of probe sequences with completely complementary sequences is obtained.

[0139] The preparation of the chip mainly uses a glass slide or a silicon wafer as a carrier, and oligonucleotide fragments or cDNA are arranged on the carrier in sequence by in-situ synthesis and microarray methods.

[0140] The chip described in this application is based on the detection of signals from hybridization of bisulfite-treated DNA sequences. Bisulfite treatment converts non-methylated cytosine into uracil, while methylated cytosine remains unchanged. Then, uracil is further converted into thymine, and finally, chip hybridization is performed. Finally, the type of added base is determined based on the fluorescence color, and thus whether the site is methylated is determined.

[0141] This application provides a method for cervical cancer screening, which includes:

[0142] detecting the methylation level of a methylation marker, and

[0143] judging the risk of a subject suffering from cervical cancer based on the methylation level, where the methylation marker is the KHDRBS2 gene.

[0144] Example

[0145] In the present invention, the materials and experimental methods used in the experiments are described generally and / or specifically. In the following examples, unless otherwise specified, % represents wt%, that is, weight percentage. Reagents or instruments without indicating the manufacturer can be obtained as conventional reagent products through commercial purchase.

[0146] Example 1 Screening of markers

[0147] 1) Sample collection: A total of 98 cervical exfoliated cell samples from early-stage cervical cancer patients were collected from a cooperative hospital, and 85 cervical exfoliated cell samples from healthy individuals were collected. DNA extraction and purification were performed (see details in 1.1).

[0148] 2) Analysis of candidate markers: The DSS software was used to perform differential analysis between healthy individuals and cervical cancer patients for the methylation sites obtained from the sequencing data. The filtering criteria were: P < 0.05, and the difference in the means of the two groups was greater than 0.1. A total of 165 differentially methylated regions were obtained.

[0149] 3) Marker verification: Probes were designed to capture the above 165 differentially methylated regions, and the data of cervical exfoliated cell samples from Borecheng (number of early-stage cervical cancer samples = 77, number of healthy samples = 83) were used for verification. Finally, 1 methylation marker that can distinguish early-stage cervical cancer from healthy individuals was obtained, and its target sequences are shown in SEQ ID NO: 1.

[0150] According to the obtained target sequence region, a probe panel is customized, which includes a first probe panel for hypermethylation and a second probe panel for hypomethylation. Among them, the first probe panel includes two probes, with nucleotide sequences shown in SEQ ID NO: 7-8 respectively, and the second probe panel contains two probes, which are shown in SEQ ID NO: 9-10 respectively. Among them, the methylation marker is the KHDRBS2 gene, the sequence of its first probe panel is shown in SEQ ID NO: 7-8, and the sequence of the second probe panel is shown in SEQ ID NO: 9-10;

[0151] The sequence of SEQ ID NO: 7 is as follows:

[0152]

[0153] The sequence of SEQ ID NO: 8 is as follows:

[0154]

[0155] The sequence of SEQ ID NO: 9 is as follows:

[0156]

[0157] The sequence of SEQ ID NO: 10 is as follows:

[0158]

[0159] Then it is verified in cervical exfoliated cell samples. In the experiment, a magnetic bead method universal genomic DNA extraction kit (DP705) is used for DNA preparation. The IDT's xGen Methyl-Seq Lib KIT is used for library preparation, and the product number is 10009824. The experimental detection method is as follows:

[0160] 1.1. DNA extraction and purification:

[0161] 1.1.1. Preparation of cervical exfoliated cell samples:

[0162] Use a speculum to dilate the vagina, expose the cervix, and scrape the cervical secretions in a circular motion at the cervical os with a special small brush and place them in the preservation solution.

[0163] 1.1.2. Lysis and binding:

[0164] 1.1.2.1. Transfer the preservation solution containing the swab sample to a 15 mL centrifuge tube, and make up to 300 μl with tissue digestion solution GHA. Add 20 μl of Proteinase K and vortex for 10 sec to mix evenly.

[0165] 1.1.2.2. Place it at 75 °C for 15 min, and invert and mix it evenly 3 times during this period, 3 - 5 times each time. Take out the centrifuge tube for subsequent experiments.

[0166] 1.1.2.3. Add 300 μl of lysis solution GHL and 300 μl of isopropanol, and shake and mix evenly.

[0167] 1.1.2.4. Add 15 μl of magnetic bead suspension GH, shake and mix evenly for 1 min, let it stand for a total of 9 min, and shake and mix evenly for 1 min every 3 min.

[0168] 1.1.3. Washing:

[0169] 1.1.3.1. Place the centrifuge tube on the magnetic rack and let it stand for 30 sec. After the magnetic beads are completely adsorbed, carefully aspirate the liquid.

[0170] 1.1.3.2. Add 900 μl of buffer GDZ (please check whether absolute ethanol has been added before use), and shake and mix evenly for 2 min.

[0171] 1.1.3.3. Place the centrifuge tube on the magnetic rack and let it stand for 30 sec. After the magnetic beads are completely adsorbed, carefully aspirate the liquid.

[0172] 1.1.3.4. Add 500 μl of buffer GDZ, and shake and mix evenly for 2 min.

[0173] 1.1.3.5. Place the centrifuge tube on the magnetic rack and let it stand for 30 sec. After the magnetic beads are completely adsorbed, carefully aspirate the liquid.

[0174] 1.1.3.6. Take the centrifuge tube off the magnetic rack, add 900 μl of wash solution PWD (please check whether absolute ethanol has been added before use), and shake and mix evenly for 2 min.

[0175] 1.1.3.7. Place the centrifuge tube on the magnetic rack and let it stand for 30 sec. After the magnetic beads are completely adsorbed, carefully aspirate the liquid

[0176] 1.1.3.8. Take the centrifuge tube off the magnetic rack, add 300 μl of wash solution PWD, and shake and mix evenly for 2 min.

[0177] 1.1.3.9. Place the centrifuge tube on the magnetic rack and let it stand for 30 sec. After the magnetic beads are completely adsorbed, carefully aspirate the liquid.

[0178] 1.1.3.10. Place the centrifuge tube on the magnetic rack and air-dry it at room temperature for 10 - 15 min.

[0179] 1.1.4. Elute DNA:

[0180] 1.1.4.1. Remove the centrifuge tube from the magnetic stand, add 50 - 100 μl of elution buffer TB, mix well by oscillation, place at 56 °C, incubate for 10 min, and invert and mix 3 times during this period, 3 - 5 times each time.

[0181] 1.1.4.2. Place the centrifuge tube on the magnetic stand and let it stand for 2 min. After the magnetic beads are completely adsorbed, carefully transfer the DNA solution to a new centrifuge tube and store it under appropriate conditions.

[0182] For DNA samples, use Agilent 2100 for fragment detection and Qubit for concentration measurement for subsequent experiments.

[0183] 1.2. Bisulfite conversion and purification:

[0184] 1.2.1. Prepare CT Conversion Reagent:

[0185] 1.2.1.1. Add 700 μl of NF water, 300 μl of M - Dilution Buffer, and 50 μl of M - Dissolving Buffer to a tube of CT conversion reagent, mix well at room temperature, and vortex or shake frequently for 10 min.

[0186] 1.2.1.2. After mixing, aliquot it to prepare enough for 10 reactions at a time.

[0187] 1.2.2. Perform bisulfite conversion on the DNA library and prepare the reaction system according to the following table.

[0188] Table 1

[0189] Component Volume of high-concentration sample (1 ng - 2 μg) Sample from the previous reaction 40 μl CT Conversion Reagent 110 μl Total volume 150 μl

[0190] Adjust the pipette to 100 μl, gently pipette and mix 6 times, then divide it into two tubes and place them on the PCR instrument.

[0191] 1.2.4. Set the following program to perform the reaction on the PCR instrument: The hot lid temperature is 105 °C.

[0192] Table 2

[0193] Temperature Time 98℃ 10 min 64℃ 2.5h 4℃ ∞

[0194] 1.2.5. Take a new 1.5 - ml centrifuge tube and add 600 μl of M - Binding Buffer.

[0195] 1.2.6. After the PCR is completed, briefly centrifuge and transfer the two tubes of the same sample to the corresponding 1.5 - ml centrifuge tubes respectively, and mix well.

[0196] 1.2.7. Add the above - mixed sample to the Zymo - Spin TM IC Column, invert to mix well, and centrifuge at 10,000 x g for 30 s.

[0197] 1.2.8. Add 100 μl of M - Wash Buffer to the column, and centrifuge at 10,000 x g for 30 s.

[0198] 1.2.9. Add 200 μl of M - Desulphonation Buffer to the column, let it stand at room temperature for 15 - 20 min, and centrifuge at 10,000 x g for 30 s.

[0199] 1.2.10. Add 200 μl of M - Wash Buffer to the column, and centrifuge at 10,000 x g for 30 s.

[0200] 1.2.11. Repeat the previous step once.

[0201] 1.2.12. Place the column in a new collection tube and centrifuge at 10,000 x g for another 30 s.

[0202] 1.2.13. Place the recovery column in a new 1.5 - ml EP tube, add 15 μl of LOW EDTA buffer to the center of the column membrane, and centrifuge at 10,000 x g for 30 s.

[0203] 1.3. Denaturation:

[0204] 1.3.1. Pre - heat the PCR instrument to 95 °C.

[0205] 1.3.2. Set the following program for reaction on the PCR instrument: The hot - lid temperature is 105 °C.

[0206] Table 3

[0207] Temperature Time 95℃ ∞ 95℃ 2 min 95℃ ∞

[0208] 1.3.3. Immediately place the test tube on ice for 2 min after incubation.

[0209] 1.4. Adapter ligation and purification:

[0210] 1.4.1. Configure the reaction system with reference to the following table:

[0211] Table 4

[0212] Component Volume Low EDTA TE 11.5 μl Buffer G1 4 μl Reagent G2 4 μl Reagent G3 2.5 μl Enzyme G4 1 μl Enzyme G5 1 μl Enzyme G6 1 μl DNA 15ul Total Volume 40ul

[0213] 1.4.2. Set the following program for reaction on the PCR instrument: The hot - lid temperature is 105 °C.

[0214] Table 5

[0215] Temperature Time 37℃ ∞ 37℃ 15 min 95℃ 2 min 4℃ ∞

[0216] 1.5. Sample Extension and Purification:

[0217] 1.5.1. Configure the reaction system according to the following table:

[0218] Table 6

[0219] Component Volume Reagem Y1 2 μl Enzyme Y2 42 μl Total Volume 44 μl

[0220] 1.5.2. Set the following program to perform the reaction on a PCR instrument: The hot lid temperature is 105°C.

[0221] Table 7

[0222] Temperature Time 98℃ ∞ 98℃ 1 min 62℃ 2 min 65℃ 5 min 4℃ ∞

[0223] 1.5.3. Add DNA protection buffer and the liquid turns blue. Gently pipette and mix, then divide into two tubes and place on the PCR instrument.

[0224] 1.5.4. Set the following program and run: Hot lid 105°C.

[0225] Table 8

[0226]

[0227]

[0228] 1.5.5. Prepare the purification system according to the following table:

[0229] Table 9

[0230] Input amount Reaction volume Amount of magnetic beads Volume 200 bp (SeqCap Epi) 84ul 101 μl (ratio: 1.2) 15 μl

[0231] 1.5.6. Add the magnetic beads in the above ratio to each sample for recovery, shake and mix well, and centrifuge briefly.

[0232] 1.5.7. Incubate at room temperature for 5 min.

[0233] 1.5.8. Shake and mix well, centrifuge briefly, place on the magnetic stand for adsorption until the solution becomes clear (~2 min), and aspirate the supernatant after the solution is clear.

[0234] 1.5.9. Add 200 μl of 80% ethanol to wash the magnetic beads for 30 s, discard the supernatant, and carefully remove all the remaining ethanol on the inner wall of the pipette.

[0235] 1.5.10. Repeat the above steps.

[0236] 1.5.11. Elute with the optimal volume of low EDTA TE buffer, then shake and mix well.

[0237] 1.5.12. Incubate at room temperature for 2 min.

[0238] 1.5.13. Place on a magnetic stand and adsorb until the solution becomes clear (~2 min). After the solution becomes clear, aspirate the supernatant.

[0239] 1.5.14. Transfer the entire eluate to a new 0.2 mL PCR tube, ensuring that the eluate does not contain magnetic beads.

[0240] 1.6. Ligation and purification of adapters:

[0241] 1.6.1. Prepare the library reaction system according to the following table:

[0242] Table 10

[0243] Component Volume Buffer B1 3 μl Reagent B2 10 μl Enzyme B3 2 μl TotalVolume 15 μl

[0244] 1.6.2. Set the following program and run: Hot lid 0℃:

[0245] Table 11

[0246]

[0247]

[0248] 1.6.3. Prepare the purification system according to the following table:

[0249] Table 12

[0250] Input amount Reaction volume Amount of magnetic beads Volume 200 bp (SeqCap Epi) 30ul 36 μl (ratio: 1.2) 20 μl

[0251] 1.6.4. Add magnetic beads in the above ratio to each sample for recovery, vortex and mix well, then centrifuge briefly.

[0252] 1.6.5. Incubate at room temperature for 5 min.

[0253] 1.6.6. Vortex and mix well, centrifuge briefly, then place on a magnetic stand and adsorb until the solution becomes clear (~2 min). After the solution becomes clear, aspirate the supernatant.

[0254] 1.6.7. Add 200 μl of 80% ethanol to wash the magnetic beads for 30 s, discard the supernatant, and carefully remove all remaining ethanol on the inner wall of the pipette.

[0255] 1.6.8. Repeat the above steps.

[0256] 1.6.9. Add the optimal volume of low EDTA TE buffer recommended in the above table for elution, then vortex and mix well.

[0257] 1.6.10. Incubate at room temperature for 2 min.

[0258] 1.6.11. Place it on the magnetic stand and adsorb until the solution becomes clear (~2 min). After the solution becomes clear, aspirate the supernatant.

[0259] 1.6.12. Transfer the entire eluate to a new 0.2 mL PCR tube, ensuring that the eluate does not contain magnetic beads.

[0260] 1.7. Library amplification and purification:

[0261] 1.7.1. Prepare the library reaction system according to the following table:

[0262] Table 13

[0263] Component Volume DNA from the above reaction 20 μl KAPA HiFi HotStart Uracil+ReadyMix (2x) 25 μl index (U001 - U024) 5 μl Total volume 50 μl

[0264] 1.7.2. Set the following program and run: Hot lid 105 °C:

[0265] Table 14

[0266]

[0267]

[0268] 1.7.3. The recommended number of cycles is as follows in the table:

[0269] Table 15

[0270] Input amount Recommended cycle number 20 ng cfDNA 10-11 100 ng gDNA 9-10 20 ng gDNA 11-12

[0271] 1.7.4. Prepare the purification system according to the following table:

[0272] Table 16

[0273] Input amount Reaction volume Amount of magnetic beads Volume 200 bp (SeqCap Epi) 50 μl 60 μl (ratio: 1.2) 22 μl

[0274] 1.7.5. Transfer the PCR product to a 1.5 ml centrifuge tube.

[0275] 1.7.6. Add magnetic beads in the above ratio to each sample for recovery, shake well and centrifuge briefly.

[0276] 1.7.7. Incubate at room temperature for 5 min.

[0277] 1.7.8. Shake well and centrifuge briefly, then place it on the magnetic stand and adsorb until the solution becomes clear (~2 min). After the solution becomes clear, aspirate the supernatant.

[0278] 1.7.9. Add 500 μl of 80% ethanol to wash the magnetic beads for 30 s, discard the supernatant, and carefully remove all the remaining ethanol on the inner wall of the pipette.

[0279] 1.7.10. Repeat the above steps.

[0280] 1.7.11. Place on the magnetic stand for 5 - 10 minutes until the beads are dry (avoid over - drying as it may lead to a reduction in DNA recovery rate).

[0281] 1.7.12. Add the optimal volume of low EDTA TE buffer recommended in the above table for elution, and then mix by shaking.

[0282] 1.7.13. Incubate at room temperature for 2 min.

[0283] 1.7.14. Place on the magnetic stand for adsorption until the solution becomes clear (~2 min). After the solution becomes clear, aspirate the supernatant.

[0284] 1.7.15. Transfer the entire eluate to a new 0.2 mL PCR tube, ensuring that the eluate does not contain magnetic beads.

[0285] 1.7.16. Aspirate 1 μL for qubit calibration and perform 2100 quality inspection.

[0286] 1.8. Sample hybridization with the probe:

[0287] 1.8.1. Mix the samples:

[0288] 1.8.1.1. Refer to the following table for the usage amount of the DNA library. The total usage amount can exceed 1500 ng but not be greater than 4 μg;

[0289] Table 17

[0290] Number of mixed samples Usage per library Total amount per reaction library 1 500 ng 500 ng 2 500 ng 1000 ng 3 500 ng 1500 ng 4 375 ng 1500 ng 8 187.5 ng 1500 ng

[0291] 1.8.1.2. Calculate the usage amounts of different samples and mix them evenly in a centrifuge tube.

[0292] 1.8.1.3. Add the following pre - hybridization reagents to the mixed samples respectively, mix well, and try not to generate bubbles.

[0293] Table 18

[0294] Component Volume Twist probe panel 4 μl Universal blocker 8 μl Blocker solution 5 μl Methylation Enhancer 2 μl

[0295] 1.8.1.4. Dry the above - mixed pre - hybridization reagents at room temperature (use low temperature if heating is required) in a vacuum concentrator.

[0296] 1.8.2. Hybridization:

[0297] 1.8.2.1. Incubate the Fast Hybridization Mix at 65 °C for 10 min or until all the precipitate dissolves. Vortex it rapidly and add 20 μl to the lyophilized sample from the previous step to resuspend the sample (do not allow the hybridization solution to return to room temperature). Gently flick it with your finger to mix evenly, avoiding the generation of bubbles.

[0298] 1.8.2.2. Centrifuge rapidly to remove bubbles, and add 30 μl of Hybridization Enhancer onto the surface of the above reagent.

[0299] 1.8.2.3. Place the PCR tube into the preheated PCR instrument for hybridization.

[0300] 1.8.2.4. Set the following program and run: Heat lid at 85 °C.

[0301] Table 19

[0302] Temperature Time 95℃ ∞ 95℃ 5 min 60℃ 15 min - 4 h

[0303] 1.8.3. Binding:

[0304] 1.8.3.1. Vortex the pre-equilibrated streptavidin magnetic beads until thoroughly mixed, and add 100 μl of the magnetic beads into a 1.5-ml centrifuge tube.

[0305] 1.8.3.2. Add 200 μl of binding buffer and pipette to mix evenly.

[0306] 1.8.3.3. Place the centrifuge tube on the magnetic stand for 1 min or until the solution becomes clear. Discard the supernatant and remove the centrifuge tube.

[0307] 1.8.3.4. Repeat the above washing steps 2 more times, for a total of 3 times.

[0308] 1.8.3.5. After the last wash, add 200 μl of binding buffer, and vortex to resuspend and mix thoroughly.

[0309] 1.8.3.6. After hybridization, open the lid of the PCR instrument and rapidly transfer all the hybridization solution to the pre-equilibrated magnetic beads.

[0310] 1.8.3.7. Mix the magnetic beads with the hybridization solution thoroughly on a shaker, rocker, or rotator at room temperature for 30 min.

[0311] 1.8.3.8. Remove the centrifuge tube from the mixer, centrifuge rapidly, and then place it on the magnetic stand for 1 min. Discard the supernatant and remove the tube.

[0312] 1.8.3.9. Add 200 μl of preheated Wash Solution 1 (Fast Wash Buffer 1) and mix well.

[0313] 1.8.3.10. Incubate at 63 °C or 65 °C for 5 min.

[0314] 1.8.3.11. Place the centrifuge tube on the magnetic stand for 1 min, discard the supernatant, and remove the tube.

[0315] 1.8.3.12. Repeat the above steps, add 200 μl of pre-warmed wash buffer 1 again, and mix well.

[0316] 1.8.3.13. Incubate at 63 °C or 65 °C for 5 min.

[0317] 1.8.3.14. Transfer the liquid to a new tube; place it on the magnetic stand for 1 min, discard the supernatant, and remove the tube.

[0318] 1.8.3.15. Add 200 μl of pre-warmed wash buffer 2, and mix well with a pipette tip.

[0319] 1.8.3.16. Incubate at 48 °C for 5 min.

[0320] 1.8.3.17. Place it on the magnetic stand for 1 min, discard the supernatant, and remove the tube.

[0321] 1.8.3.18. Repeat steps 3.15 - 3.17 twice, for a total of three times.

[0322] 1.8.3.19. For the last time, pipette out the wash buffer completely with a 10 μl pipette tip.

[0323] 1.8.3.20. Add 45 μl of water, mix well, and incubate the solution on ice.

[0324] 1.8.4. Post-capture PCR amplification, purification, and quality control:

[0325] 1.8.4.1. Set the following program and run: Hot lid at 105 °C.

[0326] Table 20

[0327]

[0328] 1.8.4.2. Mix the magnetic bead mixture from step 1.8.3.20, and pipette 22.5 μl into a 0.2 ml PCR tube.

[0329] 1.8.4.3. Add 2.5 μl of amplification primer and 25 μl of KAPA HiFi HotStart ReadyMix to the 0.2 ml PCR tube, for a total reaction volume of 50 μl.

[0330] 1.8.4.4. Gently mix with a pipette tip, centrifuge quickly, and then place it in a PCR instrument to start amplification.

[0331] 1.8.4.5. Vortex the pre-equilibrated DNA purification magnetic beads thoroughly.

[0332] 1.8.4.6. Add 90 μl (1.8*) of DNA purification magnetic beads to the amplified PCR product and vortex thoroughly to mix.

[0333] 1.8.4.7. Incubate at room temperature for 5 min.

[0334] 1.8.4.8. Place the centrifuge tube on a magnetic stand for 1 min. After the solution becomes clear, discard the supernatant.

[0335] 1.8.4.9. Without removing the centrifuge tube from the magnetic stand, directly add freshly prepared 200 μl of 80% ethanol, incubate for 1 min, and discard the supernatant; repeat the 80% ethanol wash (a total of 2 times), and keep the centrifuge tube on the magnetic stand.

[0336] 1.8.4.10. Carefully remove the residual ethanol with a 10 μl pipette tip and let it stand at room temperature for 5 - 10 min or until the magnetic beads are dry. Note that the magnetic beads should not be overdried.

[0337] 1.8.4.11. Remove the tube from the magnetic stand and add 32 μl of water. Pipette to mix thoroughly and incubate at room temperature for 2 min.

[0338] 1.8.4.12. Place the centrifuge tube on the magnetic stand for 3 min or until the solution becomes clear.

[0339] 1.8.4.13. Transfer 30 μl of the supernatant to a clean 0.2 ml centrifuge tube.

[0340] 1.8.4.14. Take 1 μl of the library and quantify it using Qubit, and record the library concentration.

[0341] 1.8.4.15. Take 1 μl of the sample and measure the library fragment length using Agilent 2100.

[0342] 1.8.4.16. Perform sequencing using the Illumina high-throughput sequencing platform.

[0343] 1.9. Bioinformatics analysis process for methylation. Generally as follows: Use the fastp quality control software to check the sequencing quality, remove low-quality reads, then use the Bismark alignment software to align the quality-controlled clean data to the reference genome, and use the Bismar_methylation_extractor software to extract the corresponding methylation sites. Finally, calculate the methylation level of the target region. If the value result exceeds the threshold, it is judged as cancer; if it is lower than the threshold, it is judged as normal.

[0344] 1.10. Based on a dataset of 98 early-stage cervical cancer samples and 85 normal samples in the training set, using the above-mentioned methylation library construction method, screen out biomarkers related to early-stage cervical cancer by using the methylation level differences in different groups (cervical cancer and normal), and verify the site data in independent datasets of normal and early-stage cervical cancer samples. One DNA fragment that most significantly differentiates normal and cancer samples is screened out. The methylation biomarker (hereinafter referred to as the site or marker) and the discrimination threshold are shown in Table 21;

[0345] The calculation method of the methylation level threshold is as follows: Draw an ROC curve based on the dataset (including 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 calculating indicators such as sensitivity, specificity, and accuracy. Usually, we will select 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 range of the Youden index is between 0 and 1, representing the total ability of the classification model to identify true patients and non-patients. The larger the Youden index, the better the performance of the classification model.

[0346] Table 21 Specific performance data of methylation markers

[0347]

[0348] Example 2

[0349] For 6 human samples (S1-3 are healthy human samples, and S4-6 are early-stage cervical cancer patient samples), use the methylation biomarker detection method of the present application. Collect cervical exfoliated cells according to the method of Example 1; construct a library and sequence through the Illumina platform; the sequencing data is analyzed through the above-mentioned bioinformatics process to obtain the methylation level of each methylation biomarker. According to the threshold of each methylation biomarker, predict the disease status of the patient. If it exceeds the threshold, it is an early-stage cervical cancer sample; if it is lower than the threshold, it is a healthy human sample. The specific results are as follows in the table:

[0350] Among the interpretation results, 0 represents being classified as normal, that is, healthy; 1 represents being classified as abnormal, that is, having a tumor.

[0351] Table 22

[0352]

[0353] In summary, the inventors of the present application have obtained methylation genes related to cervical cancer, determined the target sequences where methylation abnormalities occur in the methylation genes of cervical cancer, and through the target sequences of these methylation genes, the methylation status of the genes can be detected sensitively and specifically, so that it can be used for the detection of DNA in exfoliated cervical cells. Moreover, the composition described in the present application can achieve real-time monitoring and has higher sensitivity and accuracy.

[0354] The above are only the preferred embodiments of the present invention, and are not limitations to the present invention in other forms. Any person skilled in the art may use the technical content disclosed above to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still belong to the protection scope of the technical solution of the present invention.

Claims

1. A methylation marker for detecting cervical cancer, wherein, The methylation marker is the KHDRBS2 gene.

2. The methylation marker according to claim 1, wherein, The target sequence of the methylation marker is shown as any one of SEQ ID NOs: 1-6 or contains a sequence shown as any one of SEQ ID NOs: 1-6.

3. A probe composition, wherein, The probe composition contains a probe targeting the methylation of the target sequence of the methylation marker described in claim 1 or 2.

4. The probe composition according to claim 3, wherein, The probe composition contains a first probe composition for high methylation and a second probe composition for low methylation. The first probe composition is used to hybridize with the CG hypermethylated region after bisulfite conversion, and the second probe composition is used to hybridize with the CG hypomethylated region after bisulfite conversion; Preferably, the first probe composition includes n probes, and the n probes hybridize with each nucleotide of the sense strand and / or antisense strand of the CG hypermethylated region after bisulfite conversion. Preferably, the second probe composition includes m probes, and the m probes hybridize with each nucleotide of the sense strand and / or antisense strand of the CG hypomethylated region after bisulfite conversion. Further preferably, both n and m are any integers from 1 to 10; Preferably, there are x1 nucleotide overlaps between the (n-1)th probe and the nth probe. Preferably, x1 is any integer from 0 to 100; Preferably, there are x2 nucleotide overlaps between the (m-1)th probe and the mth probe. Preferably, x2 is any integer from 0 to 100; Further preferably, the first probe composition includes the nucleotide sequences shown in SEQ ID NO: 7-8; the second probe composition includes the nucleotide sequences shown in SEQ ID NO: 9-10.

5. Use of a nucleic acid for detecting a target sequence of a methylation marker in the preparation of a kit for detecting cervical cancer, wherein, The methylation marker is the KHDRBS2 gene.

6. The use according to claim 5, wherein The target sequence of the methylation marker is shown as any one of SEQ ID NOs: 1-6 or contains a sequence shown as any one of SEQ ID NOs: 1-6; Preferably, the nucleic acid is used to target the target sequence of the methylation marker after methylation in cervical cancer; Preferably, the nucleic acid is the probe composition described in any one of claims 3-4.

7. Use according to any one of claims 5-6, wherein The nucleic acid includes: Primers, which are fragments of at least 9 nucleotides in the target sequence of the KHDRBS2 gene, and the fragment contains at least one CpG dinucleotide sequence; Preferably, the nucleic acid further includes: Probes, which hybridize with at least 15 nucleotide fragments in the target sequence of the KHDRBS2 gene under medium stringency or stringent conditions, and the fragment contains at least one CpG dinucleotide sequence; Preferably, the nucleic acid further includes: A blocker that preferentially binds to the target sequence in the unmethylated state.

8. A composition for cervical cancer detection, wherein, The composition contains a nucleic acid for detecting the methylation of the KHDRBS2 gene.

9. The composition according to claim 8, wherein The target sequence of the KHDRBS2 gene is shown as any one of SEQ ID NOs: 1-6 or contains a sequence shown as any one of SEQ ID NOs: 1-6; Preferably, the nucleic acid contains the probe composition described in any one of claims 3-4.

10. The composition according to claim 8 or 9, wherein The nucleic acid includes: Primers, wherein the primers are fragments of at least 9 nucleotides in the target sequence of the methylation marker, and the fragments contain at least one CpG dinucleotide sequence; Preferably, the nucleic acid further comprises: Probes, wherein the probes hybridize with at least 15 nucleotide fragments in the target sequence of the methylation marker under medium stringency or stringent conditions, and the fragments contain at least one CpG dinucleotide sequence.

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