Composition for detecting ovarian cancer and use thereof

Abstract

A composition for detecting ovarian cancer and a use thereof. The composition comprises: a nucleic acid for detecting the methylation state of a target gene, the target gene being one or more of a TFAP2E gene, a WNT6 gene, an EMX2OS gene, a CRYBG1 gene, and an IFFO1 gene. A kit comprising the composition, and a use of the composition in the preparation of a kit for detecting ovarian cancer in vitro.

Description

Compositions for detecting ovarian cancer and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology, and relates to gene detection, in particular to a nucleic acid composition for detecting methylation of an ovarian cancer related gene, and a corresponding kit and use thereof. BACKGROUND

[0002] Ovarian cancer is one of the common gynecological malignancies, and its mortality rate ranks first among female reproductive tract malignancies, which is a malignant tumor seriously threatening women's health. Ovarian cancer has high heterogeneity, about 90% of which belongs to epithelial ovarian cancer, about 70% of which is serous, and the prognosis is poor. The 5-year survival rate of patients with early-stage (stage I) ovarian cancer is more than 90%, however, early-stage ovarian cancer has no obvious clinical symptoms, and most patients are difficult to be discovered in time in the early stage; 60%-70% of patients are in the late stage when diagnosed. However, the 5-year survival rate of patients with stage I ovarian cancer can be as high as 90%, about 80% for stage II, and only 30%-40% for stage III / IV. Therefore, it is of great significance to find a reliable early detection method for ovarian cancer.

[0003] Currently, there are several methods for detecting ovarian cancer: Transvaginal ultrasound (TVUS) uses ultrasound waves to transmit images that can help identify potential growths in the ovaries and determine whether they are solid growths or cysts (non-cancerous, fluid-filled sacs). If a solid mass is found, a biopsy may be required to determine whether the mass is benign or malignant. However, the disadvantage of B-ultrasound is that it is not specific, and its diagnostic accuracy is low for masses smaller than 2 cm in diameter or those that are atypical. CT scans are currently the best method for determining the location, size, extent, and nature of ovarian cancer, but they still cannot make a qualitative judgment for some tumors. The CA-125 blood test is also a common method for screening and monitoring ovarian cancer. Many ovarian cancer patients will have elevated CA-125 levels in their blood, but not all ovarian cancer patients will have elevated CA-125 levels. According to the Ovarian Cancer Research Fund Alliance (OCRFA), approximately 80% of advanced ovarian cancer patients have high CA-125 levels, while 50% of patients have high CA-125 levels at the early stage of the disease. In addition, other diseases such as pelvic inflammation and endometriosis can also cause elevated CA-125 levels in the blood. Furthermore, the CA-125 blood test has some disadvantages: 1) Lack of specificity: The specificity of the CA-125 blood test is relatively low, meaning it can produce false positive results in non-cancerous situations. Other diseases, such as endometriosis, uterine fibroids, pelvic inflammation, and some benign ovarian tumors, can also cause CA-125 to rise. 2) Inability to detect early: The CA-125 blood test is not sensitive in detecting early-stage ovarian cancer. At the early stage, ovarian cancer may not cause a significant increase in CA-125 levels, so the test has limited screening capabilities for early-stage ovarian cancer. 3) Not suitable for all populations: The CA-125 blood test is mainly suitable for patients who have been diagnosed with ovarian cancer or high-risk individuals with a family history of ovarian cancer. For low-risk individuals, such as generally healthy women, this test is not part of routine screening. 4) No definitive diagnostic value: The CA-125 blood test cannot be used as the only basis for diagnosing ovarian cancer, and other tests and evaluations are needed to make a final diagnosis. It is only used as an auxiliary tool and cannot be used alone to determine whether a person has ovarian cancer. Magnetic resonance imaging (MRI) is also a commonly used detection method and is very advantageous in observing the depth of endometrial lesions invading the muscular layer and the boundary between cervical tumors and the bladder or rectum. It plays an important role in the diagnosis and differentiation of ovarian cancer and pelvic lesions. However, MRI is more expensive than CT, and patients with intrauterine devices also need to remove them before MRI can be performed. Furthermore, it has some disadvantages: 1) High cost: MRI equipment and operation costs are high, which may limit its use for some patients. It is not suitable for early diagnosis. 2) Longer time: MRI examinations usually take a long time, and a single examination may last 30 minutes to an hour.For some patients, it can not be easy to remain still in a confined space. 3) Limitations for certain populations: Due to the use of strong magnetic fields, MRI may not be suitable for some patients with metal objects such as pacemakers, artificial joints, iron vascular clips, etc. Pregnant women should also avoid MRI examination in the early stages of pregnancy. 4) Unable to provide a definitive diagnosis: MRI can show ovarian masses and abnormalities, but cannot provide a definitive diagnosis. The final diagnosis requires a comprehensive consideration of clinical history, symptoms, other imaging examinations, and possible tissue biopsy results.

[0004] Recent studies have shown that epigenetics plays an important role in the occurrence and development of cancer. As an important mechanism of epigenetics, the regulation of DNA methylation in various cancers has been extensively studied. Research data shows that the regulation of gene methylation is related to biological mechanisms such as chromatin structure and gene expression regulation; changes in cell gene methylation occur in the early stages of tumor formation and throughout the occurrence and development of cancer; methylation of tumor suppressor genes is an important molecular mechanism for the transformation of precancerous lesion tissue into malignant tumor cells. However, there is currently a lack of detection techniques, methods, and products for detecting methylation genes in ovarian cancer. Therefore, there is a need for sensitive and specific methylation gene markers for the detection of ovarian cancer. SUMMARY

[0005] In view of the defects of the existing detection methods, the purpose of the present application is to provide a new biomolecular marker with high diagnostic value for ovarian cancer, which can realize non-invasive detection of ovarian cancer in vitro and improve the detection rate of early ovarian cancer.

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

[0007] 1. A composition for detecting ovarian cancer in vitro, the composition comprising:

[0008] a nucleic acid for detecting the methylation state of a target gene,

[0009] wherein the methylation state of the target gene is characterized by methylation of a target sequence of the target gene,

[0010] wherein the target gene is one or more of the following: TFAP2E gene, WNT6 gene, EMX2OS gene, CRYBG1 gene, and IFFO1 gene.

[0011] 2. The composition according to item 1, wherein the target gene is the TFAP2E gene, the WNT6 gene, and the IFFO1 gene; or the EMX2OS gene, the CRYBG1 gene, and the IFFO1 gene.

[0012] 3. The composition of item 1, wherein the target sequence of the TFAP2E gene is set forth in any one of SEQ ID NOs: 1-12 or the target sequence of the TFAP2E gene comprises a sequence set forth in any one of SEQ ID NOs: 1-12;

[0013] Preferably, the target sequence of the TFAP2E gene comprises a sequence set forth in any one of SEQ ID NOs: 1-4 and any one of SEQ ID NOs: 5-8, or the target sequence of the TFAP2E gene comprises a sequence set forth in any one of SEQ ID NOs: 1-4 and any one of SEQ ID NOs: 9-12, or the target sequence of the TFAP2E gene comprises a sequence set forth in any one of SEQ ID NOs: 5-8 and any one of SEQ ID NOs: 9-12, or the target sequence of the TFAP2E gene comprises a sequence set forth in any one of SEQ ID NOs: 1-4 and any one of SEQ ID NOs: 5-8 and any one of SEQ ID NOs: 9-12.

[0014] 4. The composition of item 1, wherein the target sequence of the WNT6 gene is set forth in any one of SEQ ID NOs: 13-24 or the target sequence of the WNT6 gene comprises a sequence set forth in any one of SEQ ID NOs: 13-24;

[0015] Preferably, the target sequence of the WNT6 gene comprises a sequence set forth in any one of SEQ ID NOs: 13-16 and any one of SEQ ID NOs: 17-20, or the target sequence of the WNT6 gene comprises a sequence set forth in any one of SEQ ID NOs: 13-16 and any one of SEQ ID NOs: 21-24, or the target sequence of the WNT6 gene comprises a sequence set forth in any one of SEQ ID NOs: 17-20 and any one of SEQ ID NOs: 21-24, or the target sequence of the WNT6 gene comprises a sequence set forth in any one of SEQ ID NOs: 13-16 and any one of SEQ ID NOs: 17-20 and any one of SEQ ID NOs: 21-24.

[0016] 5. The composition of item 1, wherein the target sequence of the EMX2OS gene is set forth in any one of SEQ ID NOs: 25-36 or the target sequence of the EMX2OS gene comprises a sequence set forth in any one of SEQ ID NOs: 25-36;

[0017] Preferably, the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 29-32, or the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 33-36, or the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 29-32 and any one of SEQ ID NOs: 33-36, or the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 29-32 and any one of SEQ ID NOs: 33-36.

[0018] 6. The composition of item 1, wherein the target sequence of the CRYBG1 gene is as set forth in any one of SEQ ID NOs: 37-48 or the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 37-48;

[0019] Preferably, the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 37-40 and any one of SEQ ID NOs: 41-44, or the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 37-40 and any one of SEQ ID NOs: 45-48, or the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 41-44 and any one of SEQ ID NOs: 45-48, or the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 37-40 and any one of SEQ ID NOs: 41-44 and any one of SEQ ID NOs: 45-48.

[0020] 7. The composition of item 1, wherein the target sequence of the IFFOl gene is as set forth in any one of SEQ ID NOs: 49-84 or the target sequence of the IFFOl gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-84;

[0021] Preferably, the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 53-56, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 57-60, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 53-56 and any one of SEQ ID NOs: 57-60, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 53-56 and any one of SEQ ID NOs: 57-60, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 61-64 and any one of SEQ ID NOs: 65-68, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 61-64 and any one of SEQ ID NOs: 69-72, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 65-68 and any one of SEQ ID NOs: 69-72, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 61-64 and any one of SEQ ID NOs: 65-68 and any one of SEQ ID NOs: 69-72, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 73-76 and any one of SEQ ID NOs: 77-80, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 73-76 and any one of SEQ ID NOs: 81-84, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 77-80 and any one of SEQ ID NOs: 81-84, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 73-76 and any one of SEQ ID NOs: 77-80 and any one of SEQ ID NOs: 81-84.

[0022] 8. The composition of any one of clauses 1-7, wherein the nucleic acid for detecting the methylation state of the target gene comprises:

[0023] a primer that is a fragment of at least 9 nucleotides of a target sequence of the target gene,

[0024] the fragment comprising at least one CpG dinucleotide sequence;

[0025] Preferably, the fragment of at least 9 nucleotides is a sequence as set forth in SEQ ID NO: 85 and SEQ ID NO: 86, a sequence as set forth in SEQ ID NO: 88 and SEQ ID NO: 89, a sequence as set forth in SEQ ID NO: 91 and SEQ ID NO: 92, a sequence as set forth in SEQ ID NO: 94 and SEQ ID NO: 95, a sequence as set forth in SEQ ID NO: 97 and SEQ ID NO: 98, a sequence as set forth in SEQ ID NO: 100 and SEQ ID NO: 101, a sequence as set forth in SEQ ID NO: 103 and SEQ ID NO: 104, a sequence as set forth in SEQ ID NO: 106 and SEQ ID NO: 107, a sequence as set forth in SEQ ID NO: 109 and SEQ ID NO: 110, a sequence as set forth in SEQ ID NO: 112 and SEQ ID NO: 113, a sequence as set forth in SEQ ID NO: 115 and SEQ ID NO: 116, a sequence as set forth in SEQ ID NO: 118 and SEQ ID NO: 119, a sequence as set forth in SEQ ID NO: 121 and SEQ ID NO: 122, a sequence as set forth in SEQ ID NO: 124 and SEQ ID NO: 125, a sequence as set forth in SEQ ID NO: 127 and SEQ ID NO: 128, a sequence as set forth in SEQ ID NO: 130 and SEQ ID NO: 131, a sequence as set forth in SEQ ID NO: 133 and SEQ ID NO: 134, a sequence as set forth in SEQ ID NO: 136 and SEQ ID NO: 137, a sequence as set forth in SEQ ID NO: 139 and SEQ ID NO: 140, a sequence as set forth in SEQ ID NO: 142 and SEQ ID NO: 143, or a sequence as set forth in SEQ ID NO: 145 and SEQ ID NO: 146.

[0026] 9. The composition of any one of items 1-8, wherein the nucleic acid for detecting methylation status of a target gene comprises:

[0027] a probe that hybridizes to a fragment of at least 15 nucleotides in a target sequence of the target gene under medium stringency or high stringency conditions,

[0028] the fragment comprising at least one CpG dinucleotide sequence;

[0029] Preferably, the fragment of at least 15 nucleotides is the sequence of SEQ ID NO: 87, the sequence of SEQ ID NO: 90, the sequence of SEQ ID NO: 93, the sequence of SEQ ID NO: 96, the sequence of SEQ ID NO: 99, the sequence of SEQ ID NO: 102, the sequence of SEQ ID NO: 105, the sequence of SEQ ID NO: 108, the sequence of SEQ ID NO: 111, the sequence of SEQ ID NO: 114, the sequence of SEQ ID NO: 117, the sequence of SEQ ID NO: 120, the sequence of SEQ ID NO: 123, the sequence of SEQ ID NO: 126, the sequence of SEQ ID NO: 129, the sequence of SEQ ID NO: 132, the sequence of SEQ ID NO: 135, the sequence of SEQ ID NO: 138, the sequence of SEQ ID NO: 141, the sequence of SEQ ID NO: 144, or the sequence of SEQ ID NO: 147.

[0030] 10. The composition of any one of clauses 1-9, wherein the composition further comprises:

[0031] An agent that converts a 5-position unmethylated cytosine base of a target sequence of a gene of interest to a uracil.

[0032] 11. The composition of any one of clauses 1-10, wherein the nucleic acid for detecting the methylation state of a gene of interest further comprises:

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

[0034] 12. An oligonucleotide for detecting ovarian cancer in vitro, comprising:

[0035] a fragment of at least 9 nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1-12 or a complement thereof and comprising at least one CpG dinucleotide sequence; and / or

[0036] a fragment of at least 9 nucleotides of a sequence as set forth in any one of SEQ ID NOs: 13-24 or a complement thereof and comprising at least one CpG dinucleotide sequence; and / or

[0037] a fragment of at least 9 nucleotides of a sequence as set forth in any one of SEQ ID NOs: 25-36 or a complement thereof and comprising at least one CpG dinucleotide sequence; and / or

[0038] a fragment of at least 9 nucleotides of the sequence set forth in any one of SEQ ID NOs: 37-48 or the complement thereof and comprising at least one CpG dinucleotide sequence; and / or

[0039] a fragment of at least 9 nucleotides of the sequence set forth in any one of SEQ ID NOs: 49-84 or the complement thereof and comprising at least one CpG dinucleotide sequence.

[0040] 13. The oligonucleotide according to item 12, further comprising:

[0041] a fragment of at least 15 nucleotides of the sequence set forth in any one of SEQ ID NOs: 1-12 or the complement thereof and comprising at least one CpG dinucleotide sequence; and / or

[0042] a fragment of at least 15 nucleotides of the sequence set forth in any one of SEQ ID NOs: 13-24 or the complement thereof and comprising at least one CpG dinucleotide sequence; and / or

[0043] a fragment of at least 15 nucleotides of the sequence set forth in any one of SEQ ID NOs: 25-36 or the complement thereof and comprising at least one CpG dinucleotide sequence; and / or

[0044] a fragment of at least 15 nucleotides of the sequence set forth in any one of SEQ ID NOs: 37-48 or the complement thereof and comprising at least one CpG dinucleotide sequence; and / or

[0045] a fragment of at least 15 nucleotides of the sequence set forth in any one of SEQ ID NOs: 49-84 or the complement thereof and comprising at least one CpG dinucleotide sequence.

[0046] 14. The oligonucleotide according to item 12, further comprising:

[0047] a blocker that preferentially binds to the target sequence in an unmethylated state.

[0048] 15. An oligonucleotide for use in the in vitro detection of ovarian cancer, comprising:

[0049] the sequence of SEQ ID NO: 85 and SEQ ID NO: 86, preferably further comprising the sequence of SEQ ID NO: 87; or

[0050] the sequence of SEQ ID NO: 88 and SEQ ID NO: 89, preferably further comprising the sequence of SEQ ID NO: 90; or

[0051] the sequence of SEQ ID NO: 91 and SEQ ID NO: 92, preferably further comprising the sequence of SEQ ID NO: 93; or

[0052] the sequence of SEQ ID NO: 94 and SEQ ID NO: 95, preferably further comprising the sequence of SEQ ID NO: 96; or

[0053] the sequence of SEQ ID NO: 97 and SEQ ID NO: 98, preferably further comprising the sequence of SEQ ID NO: 99; or

[0054] the sequence of SEQ ID NO: 100 and SEQ ID NO: 101, preferably further comprising the sequence of SEQ ID NO: 102; or

[0055] the sequence of SEQ ID NO: 103 and SEQ ID NO: 104, preferably further comprising the sequence of SEQ ID NO: 105; or

[0056] the sequence of SEQ ID NO: 106 and SEQ ID NO: 107, preferably further comprising the sequence of SEQ ID NO: 108; or

[0057] the sequence of SEQ ID NO: 109 and SEQ ID NO: 110, preferably further comprising the sequence of SEQ ID NO: 111 ; or

[0058] the sequence of SEQ ID NO: 112 and SEQ ID NO: 113, preferably further comprising the sequence of SEQ ID NO: 114; or

[0059] the sequence of SEQ ID NO: 115 and SEQ ID NO: 116, preferably further comprising the sequence of SEQ ID NO: 117; or

[0060] the sequence of SEQ ID NO: 118 and SEQ ID NO: 119, preferably further comprising the sequence of SEQ ID NO: 120; or

[0061] the sequence of SEQ ID NO: 121 and SEQ ID NO: 122, preferably further comprising the sequence of SEQ ID NO: 123; or

[0062] the sequence of SEQ ID NO: 124 and SEQ ID NO: 125, preferably further comprising the sequence of SEQ ID NO: 126; or

[0063] the sequence of SEQ ID NO: 127 and SEQ ID NO: 128, preferably further comprising the sequence of SEQ ID NO: 129; or

[0064] the sequence of SEQ ID NO: 130 and SEQ ID NO: 131, preferably further comprising the sequence of SEQ ID NO: 132; or

[0065] the sequence of SEQ ID NO: 133 and SEQ ID NO: 134, preferably further comprising the sequence of SEQ ID NO: 135; or

[0066] the sequence of SEQ ID NO: 136 and SEQ ID NO: 137, preferably further comprising the sequence of SEQ ID NO: 138; or

[0067] the sequence of SEQ ID NO: 139 and SEQ ID NO: 140, preferably further comprising the sequence of SEQ ID NO: 141; or

[0068] the sequence of SEQ ID NO: 142 and SEQ ID NO: 143, preferably further comprising the sequence of SEQ ID NO: 144; or

[0069] the sequence of SEQ ID NO: 145 and SEQ ID NO: 146, preferably further comprising the sequence of SEQ ID NO: 147.

[0070] 16. A kit comprising the composition of any one of items 1-11 or comprising the oligonucleotide of any one of items 12-15.

[0071] 17. The kit of item 16, further comprising at least one additional component selected from the group consisting of:

[0072] nucleotides, a DNA polymerase, and a buffer required for the function of the DNA polymerase.

[0073] 18. The kit of item 16 or 17, wherein the sample for detection comprises a cell line, a histological section, a tissue biopsy / paraffin-embedded tissue, a body fluid, a stool, a colonic effluent, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, or a combination thereof.

[0074] 19. The kit according to any one of items 16-18, further comprising: an instruction manual.

[0075] 20. Use of the composition according to any one of items 1-11 or the oligonucleotide according to any one of items 12-15 in the manufacture of a kit for in vitro detection of ovarian cancer.

[0076] 21. The use according to item 20, wherein the kit for in vitro detection of ovarian cancer detects ovarian cancer by a method comprising the steps of:

[0077] 1) isolating a DNA sample comprising a target sequence of a target gene or a fragment thereof from a biological sample to be tested;

[0078] 2) determining the methylation state of the target sequence of the target gene;

[0079] 3) determining the state of the biological sample by the detection result of the methylation state of the target sequence of the target gene, thereby achieving in vitro detection of ovarian cancer.

[0080] 22. The use according to item 21, wherein the method comprises the steps of:

[0081] extracting genomic DNA from the biological sample to be tested;

[0082] treating the extracted genomic DNA with a reagent to convert 5 un-methylated cytosine bases to uracil or other bases;

[0083] contacting the reagent-treated DNA sample with a DNA polymerase and primers for the target sequence of the target gene to perform a DNA polymerization reaction;

[0084] detecting the amplification product with a probe; and

[0085] determining the methylation state of at least one CpG dinucleotide of the target sequence of the target gene based on whether the amplification product is present.

[0086] 23. The use according to item 22, wherein the reagent is a bisulfite reagent.

[0087] 24. Use of one or more than two of a TFAP2E gene, a WNT6 gene, an EMX2OS gene, a CRYBG1 gene, and an IFF01 gene in the manufacture of a kit for in vitro detection of ovarian cancer.

[0088] 25. The use according to item 24, wherein the target sequence of the TFAP2E gene is as set forth in any one of SEQ ID NOs: 1-12 or the target sequence of the TFAP2E gene comprises a sequence as set forth in any one of SEQ ID NOs: 1-12.

[0089] Preferably, the target sequence of the TFAP2E gene comprises a sequence as set forth in any one of SEQ ID NOs: 1-4 and any one of SEQ ID NOs: 5-8, or the target sequence of the TFAP2E gene comprises a sequence as set forth in any one of SEQ ID NOs: 1-4 and any one of SEQ ID NOs: 9-12, or the target sequence of the TFAP2E gene comprises a sequence as set forth in any one of SEQ ID NOs: 5-8 and any one of SEQ ID NOs: 9-12, or the target sequence of the TFAP2E gene comprises a sequence as set forth in any one of SEQ ID NOs: 1-4 and any one of SEQ ID NOs: 5-8 and any one of SEQ ID NOs: 9-12.

[0090] 26. The use of item 24, wherein the target sequence of the WNT6 gene is as set forth in any one of SEQ ID NOs: 13-24 or the target sequence of the WNT6 gene comprises a sequence as set forth in any one of SEQ ID NOs: 13-24;

[0091] Preferably, the target sequence of the WNT6 gene comprises a sequence as set forth in any one of SEQ ID NOs: 13-16 and any one of SEQ ID NOs: 17-20, or the target sequence of the WNT6 gene comprises a sequence as set forth in any one of SEQ ID NOs: 13-16 and any one of SEQ ID NOs: 21-24, or the target sequence of the WNT6 gene comprises a sequence as set forth in any one of SEQ ID NOs: 17-20 and any one of SEQ ID NOs: 21-24, or the target sequence of the WNT6 gene comprises a sequence as set forth in any one of SEQ ID NOs: 13-16 and any one of SEQ ID NOs: 17-20 and any one of SEQ ID NOs: 21-24.

[0092] 27. The use of item 24, wherein the target sequence of the EMX2OS gene is as set forth in any one of SEQ ID NOs: 25-36 or the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 25-36,

[0093] Preferably, the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 29-32, or the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 33-36, or the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 29-32 and any one of SEQ ID NOs: 33-36, or the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 29-32 and any one of SEQ ID NOs: 33-36.

[0094] 28. The use of item 24, wherein the target sequence of the CRYBG1 gene is as set forth in any one of SEQ ID NOs: 37-48 or the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 37-48,

[0095] Preferably, the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 37-40 and any one of SEQ ID NOs: 41-44, or the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 37-40 and any one of SEQ ID NOs: 45-48, or the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 41-44 and any one of SEQ ID NOs: 45-48, or the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 37-40 and any one of SEQ ID NOs: 41-44 and any one of SEQ ID NOs: 45-48.

[0096] 29. The use of item 24, wherein the target sequence of the IFF01 gene is as set forth in any one of SEQ ID NOs: 49-84 or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-84,

[0097] Preferably, the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 53-56, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 57-60, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 53-56 and any one of SEQ ID NOs: 57-60, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 53-56 and any one of SEQ ID NOs: 57-60, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 61-64 and any one of SEQ ID NOs: 65-68, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 61-64 and any one of SEQ ID NOs: 69-72, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 65-68 and any one of SEQ ID NOs: 69-72, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 61-64 and any one of SEQ ID NOs: 65-68 and any one of SEQ ID NOs: 69-72, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 73-76 and any one of SEQ ID NOs: 77-80, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 73-76 and any one of SEQ ID NOs: 81-84, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 77-80 and any one of SEQ ID NOs: 81-84, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 73-76 and any one of SEQ ID NOs: 77-80 and any one of SEQ ID NOs: 81-84.

[0098] The present application has the following beneficial effects:

[0099] The application screens the related markers TFAP2E gene, WNT6 gene, EMX2OS gene, CRYBG1 gene and IFFO1 gene which can sensitively and specifically detect ovarian cancer, and determines the target sequence of the related marker abnormal methylation, which can sensitively and specifically detect the methylation state of the gene. The composition described in the application is used for screening of asymptomatic population in a non-invasive manner, has the characteristics of non-invasiveness, and can realize real-time monitoring.

[0100] Therefore, the application provides a composition, kit and detection method which can be used for in vitro detection of ovarian cancer, can conveniently, quickly and effectively detect ovarian cancer, and has important clinical application value. DETAILED DESCRIPTION

[0101] The application will be described in detail below. Although specific embodiments of the application are shown, it should be understood that the 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 application and to fully convey the scope of the application to those skilled in the art.

[0102] Unless otherwise defined, the implementation of the application will employ conventional molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and genetics techniques, which are within the scope of the conventional technical means in the art. Such techniques are described in detail in the literature, such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, 1984 edition); Animal Cell Culture (R.I. Freshney, 1987 edition); Methods in Enzymology series (Academic Press, Inc.); Current Protocols in Molecular Biology (F.M. Ausubel et al., 1987 edition, and regular updates); PCR: The Polymerase Chain Reaction (Mullis et al., 1994 edition). The primers, probes, blockers and kits used in the application can be prepared by using standard techniques known in the art.

[0103] Unless otherwise defined, the technical and scientific terms used in the application have the same meaning as that generally understood by those skilled in the art.

[0104] Definitions

[0105] "Pre-cancerous" in the present application means cells that are in an early stage of transformation into cancer cells or that are predisposed to transform into cancer cells. Such cells can exhibit one or more phenotypic traits characteristic of cancer cells.

[0106] "Stringent hybridization conditions" and "high stringency" in the present application refer to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids. Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids can be found in Tijssen, Techniques in Biochemistry and Molecular Biology - Hybridization of Nucleic Acids Probes, Principles and Protocols for Biomolecular Techniques. In general, stringency conditions are those in which the salt and temperature are chosen to be about 5-10°C lower than the melting point (Tm) for the specific sequence at the specified ionic strength. At 30% lower than the Tm, 50% of the probes will hybridize to their targets. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least twice the background hybridization, preferably 10 times the background hybridization. Exemplary stringency hybridization conditions are as follows: in 50% formamide, 5x SSC and 1% SDS, at 42°C, or in 5x SSC and 1% SDS at 65°C, followed by washing in 0.2x SSC and 0.1% SDS at 65°C.

[0107] Also, nucleic acids that do not hybridize under stringent conditions are still substantially similar if the polypeptide encoded by the nucleic acids is substantially similar, i.e., at least 80% homologous. In this case, typically, the nucleic acids are hybridized under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include hybridization in 40% formamide, 1 M NaCl and 1% SDS at 37°C, and washing in 1 x SSC at 45°C. Guidance in obtaining appropriate conditions of increasing stringency can be found in the present art. For PCR, temperatures of around 36°C are typically used for low stringency amplification, while annealing temperatures range from 32°C to 48°C depending on primer length. For high stringency PCR amplification, 62°C is typical, while annealing temperatures for high stringency hybridization range from 50°C to 65°C depending on primer length and specificity. Cycling conditions for low and high stringency amplification typically include: denaturation at 90-95°C for 30 seconds to 2 minutes, annealing for 30 seconds to 2 minutes, and extension at about 72°C for 1 to 2 minutes. Tools and guidance for low and high stringency amplification reactions can be found in the present art.

[0108] An "oligonucleotide" in the present application refers to a molecule composed of two or more nucleotides, preferably three or more nucleotides, the precise size of which can depend on a number of factors, which in turn are determined by the ultimate function and use of the oligonucleotide. In certain embodiments, an oligonucleotide can include a length of 10 nucleotides to 100 nucleotides. In certain embodiments, an oligonucleotide can include a length of 10 nucleotides to 30 nucleotides, or can have a length of 20 and 25 nucleotides. In some particular embodiments, oligonucleotides shorter than these lengths are also suitable.

[0109] A "primer" in the present application means an oligonucleotide, whether occurring in nature or synthesized, which, when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent, such as a DNA or RNA polymerase, and at an appropriate temperature and pH, is capable of acting as a point of initiation of synthesis of the desired extension product. The primer can be either single-stranded or double-stranded, and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The precise length of the primer will depend on many factors, including temperature, source of primer and method used. For diagnostic and prognostic applications, for example, oligonucleotide primers typically contain at least or more than about 9, 10, or 15, or 20, or 25 or more nucleotides, depending on the complexity of the target sequence, although they can contain fewer or more nucleotides. Factors involved in determining the appropriate length of a primer are well known to those skilled in the art.

[0110] A "primer pair" in the present application means a pair of primers that hybridize to opposite strands of a target DNA molecule or to regions flanking a target DNA region to be amplified.

[0111] A "primer site" in the present application means the region of a target DNA or other nucleic acid to which a primer hybridizes.

[0112] A "probe", when referring to a nucleic acid sequence, is used in its ordinary sense in the art to mean a selected nucleic acid sequence that, under specified conditions, hybridizes to a target sequence and can be used to detect the presence of that target sequence. A probe is a single- or double-stranded DNA of several tens to several hundreds or even thousands of base pairs in length that can bind (hybridize) to complementary non-labeled single-stranded DNA or RNA in a sample to be tested by means of denaturation, renaturation, and the high degree of precision of base pairing, forming a double-stranded complex (hybrid). Those skilled in the art will appreciate that in some instances a probe can also function as a primer, and a primer can also function as a probe.

[0113] "DNA methylation" in the present application refers to the addition of a methyl group to the 5 position of a cytosine (C), which is usually (but not necessarily) in the context of a CpG (cytosine followed by guanine) dinucleotide. As a relatively stable modification state, it can be inherited to the newly born daughter DNA during DNA replication under the action of DNA methyltransferase, and is an important epigenetic mechanism. When DNA methylation occurs, the methylation of the promoter region of a gene can lead to the transcriptional silencing of a tumor suppressor gene, and thus it is closely related to the occurrence of tumors. Abnormal methylation, including hypermethylation of tumor suppressor genes and DNA repair genes, hypomethylation of repetitive sequence DNA, and loss of imprinting of certain genes, is associated with the occurrence of various tumors. "Increased methylation level" or "significant methylation level" as used herein refers to the presence of at least one methylated cytosine nucleotide in a DNA sequence, wherein 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 unmethylated, and in certain embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more C can be methylated, wherein the C at these positions in the control DNA sample is unmethylated.

[0114] In embodiments, a variety of different methods can be used to detect DNA methylation alterations. Methods for detecting DNA methylation include, for example, methylation sensitive restriction endonuclease (MSRE) assays using southern or polymerase chain reaction (PCR) analysis, methylation specific or methylation sensitive PCR (MS-PCR), methylation sensitive single nucleotide primer extension (Ms-SnuPE), high resolution melting (HRM) analysis, bisulfite sequencing, pyrosequencing, methylation specific single strand conformation analysis (MS-SSCA), combined bisulfite restriction analysis (COBRA), methylation specific denaturing gradient gel electrophoresis (MS-DGGE), methylation specific melting curve analysis (MS-MCA), methylation specific denaturing high performance liquid chromatography (MS-DHPLC), methylation specific oligonucleotide (MSO). These assays can be PCR analysis, quantitative analysis using fluorescent labels, or southern blot analysis.

[0115] "Methylation assay" in the present application refers to any assay that determines the methylation state of one or more CpG dinucleotide sequences within a DNA sequence.

[0116] "Detecting" in this application means any process of observing a marker or a change in a marker (e.g., a change in methylation status of a marker or expression level of a nucleic acid or protein sequence) in a biological sample, whether or not the marker or change in marker is actually detected. In other words, the act of probing a sample for a marker or change in a marker is "detecting," even if the marker is determined to be absent or below a level of sensitivity. Detection can be quantitative, semi-quantitative, or non-quantitative observation, and can be based on comparison to one or more control samples. It will be understood that detecting ovarian cancer as disclosed herein includes detecting pre-cancerous cells that are beginning to develop into or will develop into ovarian cancer cells, or that have an increased propensity to develop into ovarian cancer cells. Detecting ovarian cancer can also include detecting a possible mortality or a possible prognosis of a disease condition.

[0117] "Identity" and "similarity" in the context of the present application refer to sequence similarity between two nucleic acid molecules. "Identity" or "similarity" can be measured by comparing the positions in each sequence that are aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. When the same or a similar amino acid (e.g., similar in steric properties or charge properties) occupies a position in the compared sequences, the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology / similarity or identity refers to the number of identical or similar amino acids at positions shared by the compared sequences as a function of the number of positions in the compared sequences. "Unrelated" or "nonhomologous" sequences share less than 40% identity, preferably less than 25% identity, with the sequences of the present application. In comparing two sequences, the presence of gaps or the presence of extra residues in one sequence relative to the other sequence also decreases the identity and homology / similarity. In specific embodiments, two or more sequences or subsequences are substantially or significantly homologous, similar or identical when, using BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or as determined by manual alignment and visual inspection, for example, as provided on-line by the National Center for Biotechnology Information (NCBI), their sequences are about 60% identical, or about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identical over a comparison window, or designated region in which the sequence is being compared. The definition also relates to, or can be used to test, the complement of a sequence. Thus, to the extent permitted by the context, a nucleotide sequence complementary to a specified target sequence or variant thereof is itself considered "similar" to the target sequence, and when referring to "similar" nucleic acid sequences, includes single-stranded sequences, their complements, duplexed strand complexes, sequences capable of encoding the same or similar polypeptide products, and any permissible variants of any of the foregoing. Situations in which similarity must be limited to analysis of a single nucleic acid strand sequence can include, for example, detection and quantitation of expression of a particular RNA sequence or coding sequence in a cell. The definition also includes sequences with deletions and / or additions as well as 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, 10, 21, 22, 23, 24, 25, or more nucleotides in length, 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 in length.

[0118] "Amplification" in the present application means the process of obtaining multiple copies from a particular locus of a nucleic acid, such as genomic DNA or cDNA. Amplification can be achieved using any of a variety of known means, including but not limited to polymerase chain reaction (PCR), transcription-based amplification, and strand displacement amplification (SDA).

[0119] "Real-time PCR based on fluorescence" in the present application means a method in which a fluorescent moiety is added to the PCR reaction system, the entire PCR process is monitored in real time using the accumulation of fluorescence signal, and finally unknown templates are quantitatively analyzed by a standard curve. In this PCR technique, there is an important concept, the cycle threshold, also known as Ct value. C stands for Cycle, and t stands for threshold. The meaning of Ct value is: the number of cycles experienced by the fluorescence signal in each reaction tube to reach the set threshold. For example, the method of setting the fluorescence threshold is as follows: the fluorescence signal of the first 15 cycles of PCR reaction is taken as the fluorescence background signal, and the default setting of the fluorescence threshold is 10 times the standard deviation of the fluorescence signal of 3-15 cycles.

[0120] "Cut off value of real-time PCR" in the present application means a critical Ct value for judging the positivity or negativity of a sample for a certain biomarker. According to certain specific real-time methods of the present application, "the critical Ct value (Cut Off value) is obtained based on statistical processing according to a certain amount of sample data", and the critical Ct value can be different according to the requirements of the required sensitivity or specificity.

[0121] "Sensitivity" in the present application means the proportion of cancer detected from a certain cancer sample, and its calculation formula is: Sensitivity = (detected cancer / all cancer). "Specificity" means the proportion of normal detected from a certain normal sample or a sample with benign disease, and its calculation formula is: Specificity = (detected negative / total negative).

[0122] A "label" or "detectable moiety" of the present application is a component that can be detected by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, useful labels include32P, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin or haptens and can be made detectable by incorporating radiolabels into peptides or by using antibodies that are specifically reactive with the peptides.

[0123] A variety of different methods can be used to detect nucleic acid molecules. Nucleic acid detection methods include, for example, PCR and nucleic acid hybridization (e.g., Southern blot, Northern blot, or in situ hybridization). In particular, oligonucleotides (e.g., oligonucleotide primers) capable of amplifying a target nucleic acid can be used in a PCR reaction. PCR methods generally include the steps of obtaining a sample, isolating nucleic acid (e.g., DNA, RNA, or both) from the sample, and contacting the nucleic acid with one or more oligonucleotide primers that specifically hybridize to the template nucleic acid under conditions that allow amplification of the template nucleic acid to occur. In the presence of the template nucleic acid, an amplification product is produced. Conditions for nucleic acid amplification and detection of amplification products are known to those of skill in the art. A variety of modifications to basic PCR technology have been developed, including, but not limited to, anchor PCR, RACE PCR, RT-PCR, and ligase chain reaction (LCR). Primer pairs in amplification reactions must anneal to opposite strands of the template nucleic acid and should be held at an appropriate distance from one another so that a polymerase can efficiently polymerize across the region and so that the amplification product can be readily detected, e.g., using electrophoresis. For example, computer programs such as OLIGO (Molecular Biology Insights Inc., Cascade, Colo.) can be used to design oligonucleotide primers to aid in the design of primers with similar melting temperatures. Typically, oligonucleotide primers are 9-30 or 40 or 50 nucleotides in length (e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length), although oligonucleotide primers can be longer or shorter, provided that appropriate amplification conditions are used.

[0124] Detection of amplification products or hybridization complexes is typically accomplished using a detectable label. The term "label", when referring to a nucleic acid, is intended to include direct labeling of a nucleic acid by coupling (i.e., physically linking) a detectable substance to the nucleic acid, as well as indirect labeling of the nucleic acid by reactivity with another reagent that is directly labeled with a detectable substance. Detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic groups include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin. An example of indirect labeling includes terminal labeling of a nucleic acid with biotin so that the nucleic acid can be detected with fluorescently labeled avidin.

[0125] SUMMARY

[0126] Liquid biopsy technology is to take blood, saliva, urine and other body fluids as test materials, and take tumor markers as detection indicators to realize early screening and diagnosis of cancer, auxiliary staging, prognosis and recurrence monitoring, drug guidance, etc. It has the advantages of non-invasiveness, high efficiency and accuracy. Among them, the abnormal change of DNA methylation level as a tumor molecular diagnosis marker is one of the current research hotspots and gradually becomes the consensus of the scientific and medical communities. DNA methylation is an important epigenetic modification and is involved in the regulation of various cellular processes, including embryonic development, gene transcription, X chromosome inactivation, genomic imprinting, chromatin structure stability, etc. Therefore, DNA methylation abnormalities are closely related to the occurrence of complex human diseases. In normal cells, cytosine in CpG islands and some CG-rich sites is usually non-methylated, and cytosine bases in regions with low CG proportion are mostly in a highly methylated state. However, in various cancers, the methylation pattern is exactly the opposite. Many studies have shown that high methylation of CpG islands can inhibit or silence the expression of some tumor suppressor genes and DNA mismatch repair genes, while low methylation of other regions of the genome can promote the expression of proto-oncogenes, which can give normal cells carcinogenic properties and promote the occurrence of cancer. In addition, abnormal DNA methylation usually occurs in the super-early stage of cancer and is a "seed" factor for tumor growth, and as the cancer progresses, the methylation state of DNA also changes dynamically, which can directly reflect the growth of tumor lesions. Therefore, using DNA methylation detection for early screening and auxiliary diagnosis of cancer has great application potential.

[0127] In one aspect, the present application provides a composition for detecting ovarian cancer in vitro, the composition comprising a nucleic acid for detecting methylation status within a target sequence of a target gene, wherein the target gene methylation status is characterized by methylation of the target sequence of the target gene, and wherein the target gene is one or more than two of a TFAP2E gene, a WNT6 gene, an EMX2OS gene, a CRYBG1 gene, and an IFFO1 gene.

[0128] The present application provides a group of target sequences of target genes abnormally methylated in ovarian cancer, including target sequences of one or more than two of a TFAP2E gene, a WNT6 gene, an EMX2OS gene, a CRYBG1 gene, and an IFFO1 gene, the target sequence of the TFAP2E gene being as shown in any one of SEQ ID NOs: 1-12 or comprising a sequence as shown in any one of SEQ ID NOs: 1-12, the target sequence of the WNT6 gene being as shown in any one of SEQ ID NOs: 13-24 or comprising a sequence as shown in any one of SEQ ID NOs: 13-24, the target sequence of the EMX2OS gene being as shown in any one of SEQ ID NOs: 25-36 or comprising a sequence as shown in any one of SEQ ID NOs: 25-36, the target sequence of the CRYBG1 gene being as shown in any one of SEQ ID NOs: 37-48 or comprising a sequence as shown in any one of SEQ ID NOs: 37-48, and the target sequence of the IFFO1 gene being as shown in any one of SEQ ID NOs: 49-84 or comprising a sequence as shown in any one of SEQ ID NOs: 49-84.

[0129] In one specific embodiment, the target sequence of the TFAP2E gene is as shown in SEQ ID NO: 1 (TFAP2E-1).

[0130] In one specific embodiment, the target sequence of the TFAP2E gene is as shown in SEQ ID NO: 5 (TFAP2E-2).

[0131] In one specific embodiment, the target sequence of the TFAP2E gene is as shown in SEQ ID NO: 9 (TFAP2E-3).

[0132] In one specific embodiment, the target sequence of the WNT6 gene is as shown in SEQ ID NO: 13 (WNT6-1).

[0133] In one specific embodiment, the target sequence of the WNT6 gene is as shown in SEQ ID NO: 17 (WNT6-2).

[0134] In a specific embodiment, the target sequence of the WNT6 gene is set out in SEQ ID NO: 21 (WNT6-3).

[0135] In a specific embodiment, the target sequence of the EMX2OS gene is set out in SEQ ID NO: 25 (EMX2OS-1).

[0136] In a specific embodiment, the target sequence of the EMX2OS gene is set out in SEQ ID NO: 29 (EMX2OS-2).

[0137] In a specific embodiment, the target sequence of the EMX2OS gene is set out in SEQ ID NO: 33 (EMX2OS-3).

[0138] In a specific embodiment, the target sequence of the CRYBG1 gene is set out in SEQ ID NO: 37 (CRYBG1-1).

[0139] In a specific embodiment, the target sequence of the CRYBG1 gene is set out in SEQ ID NO: 41 (CRYBG1-2).

[0140] In a specific embodiment, the target sequence of the CRYBG1 gene is set out in SEQ ID NO: 45 (CRYBG1-3).

[0141] In a specific embodiment, the target sequence of the IFF01 gene is set out in SEQ ID NO: 49 (IFF01-1).

[0142] In a specific embodiment, the target sequence of the IFF01 gene is set out in SEQ ID NO: 53 (IFF01-2).

[0143] In a specific embodiment, the target sequence of the IFF01 gene is set out in SEQ ID NO: 57 (IFF01-3).

[0144] In a specific embodiment, the target sequence of the IFF01 gene is set out in SEQ ID NO: 61 (IFF01-4).

[0145] In a specific embodiment, the target sequence of the IFF01 gene is set out in SEQ ID NO: 65 (IFF01-5).

[0146] In a specific embodiment, the target sequence of the IFF01 gene is set out in SEQ ID NO: 69 (IFF01-6).

[0147] In a specific embodiment, the target sequence of the IFF01 gene is set forth in SEQ ID NO: 73 (IFF01-7).

[0148] In a specific embodiment, the target sequence of the IFF01 gene is set forth in SEQ ID NO: 77 (IFF01-8).

[0149] In a specific embodiment, the target sequence of the IFF01 gene is set forth in SEQ ID NO: 81 (IFF01-9).

[0150] It is also understood by those skilled in the art that the target sequences of the TFAP2E gene, the WNT6 gene, the EMX2OS gene, the CRYBG1 gene, the IFF01 gene are not limited to the specific sequences listed above. The target sequences of the TFAP2E gene should cover sequences comprising one or two or three or more nucleotide mutations compared to the sequence shown in any one of SEQ ID NOs: 1-12, but substantially still have the same essential function as the sequence shown in any one of SEQ ID NOs: 1-12, also cover sequences having 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence shown in any one of SEQ ID NOs: 1-12, also cover sequences having 90%, 91%, 92%, 93%, 94%, 95% or 96% or 97% or 98% or 99% identity to the nucleotide sequence shown in any one of SEQ ID NOs: 1-12 based on deletion of one or more nucleotides, addition of one or more nucleotides, or substitution of one or more nucleotides from the nucleotide sequence shown in any one of SEQ ID NOs: 1-12. The target sequences of the WNT6 gene should cover sequences comprising one or two or three or more nucleotide mutations compared to the sequence shown in any one of SEQ ID NOs: 13-24, but substantially still have the same essential function as the sequence shown in any one of SEQ ID NOs: 13-24, also cover sequences having 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence shown in any one of SEQ ID NOs: 13-24, also cover sequences having 90%, 91%, 92%, 93%, 94%, 95% or 96% or 97% or 98% or 99% identity to the nucleotide sequence shown in any one of SEQ ID NOs: 13-24 based on deletion of one or more nucleotides, addition of one or more nucleotides, or substitution of one or more nucleotides from the nucleotide sequence shown in any one of SEQ ID NOs: 13-24. The target sequences of the EMX2OS gene should cover sequences comprising one or two or three or more nucleotide mutations compared to the sequence shown in any one of SEQ ID NOs: 25-36, but substantially still have the same essential function as the sequence shown in any one of SEQ ID NOs: 25-36, also cover sequences having 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence shown in any one of SEQ ID NOs: 25-36, also cover sequences having 90%, 91%, 92%, 93%, 94%, 95% or 96% or 97% or 98% or 99% identity to the nucleotide sequence shown in any one of SEQ ID NOs: 25-36 based on deletion of one or more nucleotides, addition of one or more nucleotides, or substitution of one or more nucleotides from the nucleotide sequence shown in any one of SEQ ID NOs: 25-36.The target sequence of the CRYBG1 gene should cover a sequence comprising one or two or three or more nucleotide mutations compared to the sequence shown in any one of SEQ ID NOs: 37-48, but substantially still the same in essential function, also cover a sequence having 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence shown in any one of SEQ ID NOs: 37-48, also cover a sequence in which one or more nucleotides are deleted, one or more nucleotides are added, or one or more nucleotides are replaced based on the nucleotide sequence shown in any one of SEQ ID NOs: 37-48, but having 90%, 91%, 92%, 93%, 94%, 95% or 96% or 97% or 98% or 99% identity to the nucleotide sequence shown in any one of SEQ ID NOs: 37-48. The target sequence of the IFF01 gene should cover a sequence comprising one or two or three or more nucleotide mutations compared to the sequence shown in any one of SEQ ID NOs: 49-84, but substantially still the same in essential function, also cover a sequence having 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence shown in any one of SEQ ID NOs: 49-84, also cover a sequence in which one or more nucleotides are deleted, one or more nucleotides are added, or one or more nucleotides are replaced based on the nucleotide sequence shown in any one of SEQ ID NOs: 49-84, but having 90%, 91%, 92%, 93%, 94%, 95% or 96% or 97% or 98% or 99% identity to the nucleotide sequence shown in any one of SEQ ID NOs: 49-84.

[0151] The target sequence of the TFAP2E gene (TFAP2E-1) (5'-3') is as follows:

[0152] The complementary sequence of the target sequence of the TFAP2E gene (TFAP2E-1) (5'-3') is as follows:

[0153] The sequence of the target sequence of the TFAP2E gene (TFAP2E-1) after bisulfite treatment (5'-3') is as follows:

[0154] The complementary sequence of the target sequence of the TFAP2E gene (TFAP2E-1) after bisulfite treatment (5'-3') is as follows:

[0155] The target sequence of the TFAP2E gene (TFAP2E-2) (5'-3') is as follows:

[0156] The complement sequence (5'-3') of the target sequence of the TFAP2E gene (TFAP2E-2) is as follows:

[0157] The sequence after bisulfite treatment (5'-3') of the target sequence of the TFAP2E gene (TFAP2E-2) is as follows:

[0158] The sequence after bisulfite treatment (5'-3') of the complement sequence of the target sequence of the TFAP2E gene (TFAP2E-2) is as follows:

[0159] The target sequence (5'-3') of the TFAP2E gene (TFAP2E-3) is as follows:

[0160] The complement sequence (5'-3') of the target sequence of the TFAP2E gene (TFAP2E-3) is as follows:

[0161] The sequence after bisulfite treatment (5'-3') of the target sequence of the TFAP2E gene (TFAP2E-3) is as follows:

[0162] The sequence after bisulfite treatment (5'-3') of the complement sequence of the target sequence of the TFAP2E gene (TFAP2E-3) is as follows:

[0163] The target sequence (5'-3') of the WNT6 gene (WNT6-1) is as follows:

[0164] The complement sequence (5'-3') of the target sequence of the WNT6 gene (WNT6-1) is as follows:

[0165] The sequence after bisulfite treatment (5'-3') of the target sequence of the WNT6 gene (WNT6-1) is as follows:

[0166] The sequence after bisulfite treatment (5'-3') of the complement sequence of the target sequence of the WNT6 gene (WNT6-1) is as follows:

[0167] The target sequence (5'-3') of the WNT6 gene (WNT6-2) is as follows:

[0168] The complement sequence (5'-3') of the target sequence of the WNT6 gene (WNT6-2) is as follows:

[0169] The target sequence of the WNT6 gene (WNT6-2) after bisulfite treatment (5'-3') is as follows:

[0170] The complementary sequence of the target sequence of the WNT6 gene (WNT6-2) after bisulfite treatment (5'-3') is as follows:

[0171] The target sequence of the WNT6 gene (WNT6-3) (5'-3') is as follows:

[0172] The complementary sequence of the target sequence of the WNT6 gene (WNT6-3) (5'-3') is as follows:

[0173] The target sequence of the WNT6 gene (WNT6-3) after bisulfite treatment (5'-3') is as follows:

[0174] The complementary sequence of the target sequence of the WNT6 gene (WNT6-3) after bisulfite treatment (5'-3') is as follows:

[0175] The target sequence of the EMX2OS gene (EMX2OS-1) (5'-3') is as follows:

[0176] The complementary sequence of the target sequence of the EMX2OS gene (EMX2OS-1) (5'-3') is as follows:

[0177] The target sequence of the EMX2OS gene (EMX2OS-1) after bisulfite treatment (5'-3') is as follows:

[0178] The complementary sequence of the target sequence of the EMX2OS gene (EMX2OS-1) after bisulfite treatment (5'-3') is as follows:

[0179] The target sequence of the EMX2OS gene (EMX2OS-2) (5'-3') is as follows:

[0180] The complementary sequence of the target sequence of the EMX2OS gene (EMX2OS-2) (5'-3') is as follows:

[0181] The target sequence of the EMX2OS gene (EMX2OS-2) after bisulfite treatment is as follows (5'-3'):

[0182] The complementary sequence of the target sequence of the EMX2OS gene (EMX2OS-2) after bisulfite treatment is as follows (5'-3'):

[0183] The target sequence of the EMX2OS gene (EMX2OS-3) is as follows (5'-3'):

[0184] The complementary sequence of the target sequence of the EMX2OS gene (EMX2OS-3) is as follows (5'-3'):

[0185] The target sequence of the EMX2OS gene (EMX2OS-3) after bisulfite treatment is as follows (5'-3'):

[0186] The complementary sequence of the target sequence of the EMX2OS gene (EMX2OS-3) after bisulfite treatment is as follows (5'-3'):

[0187] The target sequence of the CRYBG1 gene (CRYBG1-1) is as follows (5'-3'):

[0188] The complementary sequence of the target sequence of the CRYBG1 gene (CRYBG1-1) is as follows (5'-3'):

[0189] The target sequence of the CRYBG1 gene (CRYBG1-1) after bisulfite treatment is as follows (5'-3'):

[0190] The complementary sequence of the target sequence of the CRYBG1 gene (CRYBG1-1) after bisulfite treatment is as follows (5'-3'):

[0191] The target sequence of the CRYBG1 gene (CRYBG1-2) is as follows (5'-3'):

[0192] The complementary sequence of the target sequence of the CRYBG1 gene (CRYBG1-2) is as follows (5'-3'):

[0193] The sequence of the target sequence of the CRYBG1 gene (CRYBG1-2) after bisulfite treatment (5'-3') is as follows:

[0194] The sequence of the complementary sequence of the target sequence of the CRYBG1 gene (CRYBG1-2) after bisulfite treatment (5'-3') is as follows:

[0195] The sequence of the target sequence of the CRYBG1 gene (CRYBG1-3) (5'-3') is as follows:

[0196] The sequence of the complementary sequence of the target sequence of the CRYBG1 gene (CRYBG1-3) (5'-3') is as follows:

[0197] The sequence of the target sequence of the CRYBG1 gene (CRYBG1-3) after bisulfite treatment (5'-3') is as follows:

[0198] The sequence of the complementary sequence of the target sequence of the CRYBG1 gene (CRYBG1-3) after bisulfite treatment (5'-3') is as follows:

[0199] The sequence of the target sequence of the IFFO1 gene (IFFO1-1) (5'-3') is as follows:

[0200] The sequence of the complementary sequence of the target sequence of the IFFO1 gene (IFFO1-1) (5'-3') is as follows:

[0201] The sequence of the target sequence of the IFFO1 gene (IFFO1-1) after bisulfite treatment (5'-3') is as follows:

[0202] The sequence of the complementary sequence of the target sequence of the IFFO1 gene (IFFO1-1) after bisulfite treatment (5'-3') is as follows:

[0203] The sequence of the target sequence of the IFFO1 gene (IFFO1-2) (5'-3') is as follows:

[0204] The sequence of the complementary sequence of the target sequence of the IFFO1 gene (IFFO1-2) (5'-3') is as follows:

[0205] The target sequence of the IFFO1 gene (IFFO1-2) after bisulfite treatment (5'-3') is as follows:

[0206] The complementary sequence of the target sequence of the IFFO1 gene (IFFO1-2) after bisulfite treatment (5'-3') is as follows:

[0207] The target sequence (5'-3') of the IFFO1 gene (IFFO1-3) is as follows:

[0208] The complementary sequence (5'-3') of the target sequence of the IFFO1 gene (IFFO1-3) is as follows:

[0209] The target sequence of the IFFO1 gene (IFFO1-3) after bisulfite treatment (5'-3') is as follows:

[0210] The complementary sequence of the target sequence of the IFFO1 gene (IFFO1-3) after bisulfite treatment (5'-3') is as follows:

[0211] The target sequence (5'-3') of the IFFO1 gene (IFFO1-4) is as follows:

[0212] The complementary sequence (5'-3') of the target sequence of the IFFO1 gene (IFFO1-4) is as follows:

[0213] The target sequence of the IFFO1 gene (IFFO1-4) after bisulfite treatment (5'-3') is as follows:

[0214] The complementary sequence of the target sequence of the IFFO1 gene (IFFO1-4) after bisulfite treatment (5'-3') is as follows:

[0215] The target sequence (5'-3') of the IFFO1 gene (IFFO1-5) is as follows:

[0216] The complementary sequence (5'-3') of the target sequence of the IFFO1 gene (IFFO1-5) is as follows:

[0217] The target sequence of the IFFO1 gene (IFFO1-5) after bisulfite treatment (5'-3') is as follows:

[0218] The sequence (5'-3') of the complementary sequence of the target sequence of the IFF01 gene (IFF01-5) after bisulfite treatment is as follows:

[0219] The sequence (5'-3') of the target sequence of the IFF01 gene (IFF01-6) is as follows:

[0220] The sequence (5'-3') of the complementary sequence of the target sequence of the IFF01 gene (IFF01-6) is as follows:

[0221] The sequence (5'-3') of the target sequence of the IFF01 gene (IFF01-6) after bisulfite treatment is as follows:

[0222] The sequence (5'-3') of the complementary sequence of the target sequence of the IFF01 gene (IFF01-6) after bisulfite treatment is as follows:

[0223] The sequence (5'-3') of the target sequence of the IFF01 gene (IFF01-7) is as follows:

[0224] The sequence (5'-3') of the complementary sequence of the target sequence of the IFF01 gene (IFF01-7) is as follows:

[0225] The sequence (5'-3') of the target sequence of the IFF01 gene (IFF01-7) after bisulfite treatment is as follows:

[0226] The sequence (5'-3') of the complementary sequence of the target sequence of the IFF01 gene (IFF01-7) after bisulfite treatment is as follows:

[0227] The sequence (5'-3') of the target sequence of the IFF01 gene (IFF01-8) is as follows:

[0228] The sequence (5'-3') of the complementary sequence of the target sequence of the IFF01 gene (IFF01-8) is as follows:

[0229] The sequence (5'-3') of the target sequence of the IFF01 gene (IFF01-8) after bisulfite treatment is as follows:

[0230] The sequence of the complement of the target sequence of the IFF01 gene (IFF01-8) after bisulfite treatment (5'-3') is as follows:

[0231] The target sequence of the IFF01 gene (IFF01-9) (5'-3') is as follows:

[0232] The sequence of the complement of the target sequence of the IFF01 gene (IFF01-9) (5'-3') is as follows:

[0233] The sequence of the target sequence of the IFF01 gene (IFF01-9) after bisulfite treatment (5'-3') is as follows:

[0234] The sequence of the complement of the target sequence of the IFF01 gene (IFF01-9) after bisulfite treatment (5'-3') is as follows:

[0235] The target sequence and related sequences of the TFAP2E gene, the WNT6 gene, the EMX2OS gene, the CRYBG1 gene, and the IFF01 gene are shown in Table 1:

[0236] Table 1: Target sequence and related sequences of each gene

[0237] Preferably, the nucleic acid used for detecting the methylation state of the target gene comprises a fragment of at least 9 nucleotides of the target sequence of the target gene, wherein the fragment comprises at least one CpG dinucleotide sequence. In certain preferred embodiments, such as when the DNA of the sample to be tested is subjected to bisulfite conversion, the nucleic acid used for detecting the methylation state of the target gene comprises a fragment of at least 9 nucleotides of the sequence of the target sequence of the target gene after bisulfite conversion, preferably a fragment of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more nucleotides, wherein the fragment of nucleotides comprises at least one CpG dinucleotide sequence.

[0238] More preferably, the nucleic acid for detecting the methylation status of the target gene comprises a fragment of at least 15 nucleotides in the target sequence of the target gene hybridized under medium stringency or stringent conditions, wherein the fragment of nucleotides comprises at least one CpG dinucleotide sequence. In some preferred embodiments, the nucleic acid for detecting the methylation status of the target gene comprises a fragment of at least 15 nucleotides in the sequence of the target sequence of the target gene after bisulfite conversion, preferably a fragment of at least 16, 17, 18, 19, 20, 21, 22 or more nucleotides, hybridized under medium stringency or stringent conditions, wherein the fragment of nucleotides comprises at least one CpG dinucleotide sequence, if the DNA of the sample to be tested is converted by bisulfite.

[0239] Preferably, the composition further comprises an agent for converting the 5- unmethylated cytosine base of the target sequence of the target gene into uracil. More preferably, the agent is bisulfite.

[0240] The nucleic acid for detecting the methylation status of the target gene can further comprise a blocking agent that preferentially binds to DNA in the unmethylated state.

[0241] Preferably, the composition comprises one or more of the primers, probes as shown in Table 2:

[0242] Table 2 Sequences of primers and probes used in the present application

[0243] "F" in Table 2 represents a forward primer; "R" represents a reverse primer; and "P" represents a probe.

[0244] Preferably, the fluorescent labeling mode of the probe sequence used in the present application is as shown in Table 3.

[0245] Table 3 Fluorescent labeling mode of the probe sequence used in the present application

[0246] In certain embodiments, the composition further comprises an agent that converts 5- position unmethylated cytosine bases of the gene to uracil. Preferably, the agent is bisulfite. Bisulfite modification of DNA is a known tool for assessing CpG methylation status. In the DNA of eukaryotic cells, 5-methylcytosine is the most common covalent base modification. 5-methylcytosine cannot be identified by sequencing, because 5-methylcytosine has the same base pairing behavior as cytosine. Moreover, the epigenetic information carried by 5-methylcytosine is completely lost during PCR amplification. The most commonly used method for analyzing the presence of 5-methylcytosine in DNA is based on the specific reaction of bisulfite with cytosine; after subsequent alkaline hydrolysis, the unmethylated cytosine is converted to uracil, which corresponds to thymine in pairing behavior; but 5-methylcytosine remains unmodified under these conditions. The original DNA is thus converted in this way, so that the 5-methylcytosine, which originally could not be distinguished from cytosine in its hybridization behavior, is now detectable as the only remaining cytosine by conventional known molecular biology techniques, for example by amplification and hybridization. All these techniques are based on different base pairing properties, which can now be fully exploited. Thus, typically, the present application provides the use of bisulfite technology in combination with one or more methylation assays for determining the methylation status of a CpG dinucleotide sequence within a target sequence of a gene of interest. Furthermore, the methods of the present application are suitable for analyzing heterogeneous biological samples, for example low concentrations of tumor cells in blood or stool. Thus, when analyzing the methylation status of a CpG dinucleotide sequence in such a sample, the skilled person can use quantitative assays to determine the methylation level (e.g. percentage, fraction, ratio, proportion or extent) of a particular CpG dinucleotide sequence, rather than the methylation status. Accordingly, the term methylation status or methylation state should also be considered to refer to a value that reflects the methylation status of a CpG dinucleotide sequence.

[0247] In another aspect, the present application provides oligonucleotides for detecting ovarian cancer in vitro, comprising: a fragment of at least 9 nucleotides from the sequence set forth in any one of SEQ ID NOs: 1-12 or the complement thereof and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 9 nucleotides from the sequence set forth in any one of SEQ ID NOs: 13-24 or the complement thereof and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 9 nucleotides from the sequence set forth in any one of SEQ ID NOs: 25-36 or the complement thereof and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 9 nucleotides from the sequence set forth in any one of SEQ ID NOs: 37-48 or the complement thereof and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 9 nucleotides from the sequence set forth in any one of SEQ ID NOs: 49-84 or the complement thereof and comprising at least one CpG dinucleotide sequence.

[0248] Preferably the oligonucleotides for detecting ovarian cancer in vitro comprise: a fragment of at least 9 nucleotides from the sequence after bisulfite conversion of the sequence set forth in any one of SEQ ID NOs: 1-12 or the complement thereof; and / or a fragment of at least 9 nucleotides from the sequence after bisulfite conversion of the sequence set forth in any one of SEQ ID NOs: 13-24 or the complement thereof and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 9 nucleotides from the sequence after bisulfite conversion of the sequence set forth in any one of SEQ ID NOs: 25-36 or the complement thereof and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 9 nucleotides from the sequence after bisulfite conversion of the sequence set forth in any one of SEQ ID NOs: 37-48 or the complement thereof and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 9 nucleotides from the sequence after bisulfite conversion of the sequence set forth in any one of SEQ ID NOs: 49-84 or the complement thereof and comprising at least one CpG dinucleotide sequence.

[0249] The oligonucleotide for in vitro detection of ovarian cancer according to the present application can further comprise: a fragment of at least 15 nucleotides hybridizing to the sequence set forth in any one of SEQ ID NOs: 1-12 or the complement thereof under medium stringency or high stringency conditions and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 15 nucleotides hybridizing to the sequence set forth in any one of SEQ ID NOs: 13-24 or the complement thereof under medium stringency or high stringency conditions and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 15 nucleotides hybridizing to the sequence set forth in any one of SEQ ID NOs: 25-36 or the complement thereof under medium stringency or high stringency conditions and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 15 nucleotides hybridizing to the sequence set forth in any one of SEQ ID NOs: 37-48 or the complement thereof under medium stringency or high stringency conditions and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 15 nucleotides hybridizing to the sequence set forth in any one of SEQ ID NOs: 49-84 or the complement thereof under medium stringency or high stringency conditions and comprising at least one CpG dinucleotide sequence.

[0250] Preferably the oligonucleotide for in vitro detection of ovarian cancer comprises: a fragment of at least 15 nucleotides hybridizing to the sequence set forth in any one of SEQ ID NOs: 1-12 or the complement thereof after bisulfite conversion under medium stringency or high stringency conditions and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 15 nucleotides hybridizing to the sequence set forth in any one of SEQ ID NOs: 13-24 or the complement thereof after bisulfite conversion under medium stringency or high stringency conditions and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 15 nucleotides hybridizing to the sequence set forth in any one of SEQ ID NOs: 25-36 or the complement thereof after bisulfite conversion under medium stringency or high stringency conditions and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 15 nucleotides hybridizing to the sequence set forth in any one of SEQ ID NOs: 37-48 or the complement thereof after bisulfite conversion under medium stringency or high stringency conditions and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 15 nucleotides hybridizing to the sequence set forth in any one of SEQ ID NOs: 49-84 or the complement thereof after bisulfite conversion under medium stringency or high stringency conditions and comprising at least one CpG dinucleotide sequence.

[0251] The oligonucleotide for in vitro detection of ovarian cancer according to the present application can further comprise: a blocker which preferentially binds to DNA in an unmethylated state.

[0252] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 85 and SEQ ID NO: 86. It also comprises the sequence of SEQ ID NO: 87.

[0253] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 88 and SEQ ID NO: 89. It also comprises the sequence of SEQ ID NO: 90.

[0254] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 91 and SEQ ID NO: 92. It also comprises the sequence of SEQ ID NO: 93.

[0255] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 94 and SEQ ID NO: 95. It also comprises the sequence of SEQ ID NO: 96.

[0256] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 97 and SEQ ID NO: 98. It also comprises the sequence of SEQ ID NO: 99.

[0257] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 100 and SEQ ID NO: 101. It also comprises the sequence of SEQ ID NO: 102.

[0258] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 103 and SEQ ID NO: 104. It also comprises the sequence of SEQ ID NO: 105.

[0259] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 106 and SEQ ID NO: 107. It also comprises the sequence of SEQ ID NO: 108.

[0260] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 109 and SEQ ID NO: 110. It also comprises the sequence of SEQ ID NO: 111.

[0261] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 112 and SEQ ID NO: 113. It also comprises the sequence of SEQ ID NO: 114.

[0262] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 115 and SEQ ID NO: 116. It also comprises the sequence of SEQ ID NO: 117.

[0263] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 118 and SEQ ID NO: 119. It also comprises the sequence of SEQ ID NO: 120.

[0264] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 121 and SEQ ID NO: 122. It also comprises the sequence of SEQ ID NO: 123.

[0265] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 124 and SEQ ID NO: 125. It also comprises the sequence of SEQ ID NO: 126.

[0266] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 127 and SEQ ID NO: 128. It also comprises the sequence of SEQ ID NO: 129.

[0267] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 130 and SEQ ID NO: 131. It also comprises the sequence of SEQ ID NO: 132.

[0268] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 133 and SEQ ID NO: 134. It also comprises the sequence of SEQ ID NO: 135.

[0269] In a specific embodiment, an oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 136 and SEQ ID NO: 137. It also comprises the sequence of SEQ ID NO: 138.

[0270] In one specific embodiment, the oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 139 and SEQ ID NO: 140. It also comprises the sequence of SEQ ID NO: 141.

[0271] In one specific embodiment, the oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 142 and SEQ ID NO: 143. It also comprises the sequence of SEQ ID NO: 144.

[0272] In one specific embodiment, the oligonucleotide for in vitro detection of ovarian cancer comprises the sequences of SEQ ID NO: 145 and SEQ ID NO: 146. It also comprises the sequence of SEQ ID NO: 147.

[0273] In another aspect, the present application provides a kit comprising the composition. The kit also comprises at least one other component selected from the group consisting of nucleoside triphosphates, DNA polymerase and buffers required for the function of the DNA polymerase.

[0274] Typically, the kit also comprises a container for holding a patient's biological sample. Also, the kit also comprises instructions for using and interpreting the test results.

[0275] The present application also relates to the use of the above-mentioned composition and oligonucleotide in the preparation of a kit for in vitro detection of ovarian cancer.

[0276] The present application also relates to the use of one or more of the TFAP2E gene, WNT6 gene, EMX2OS gene, CRYBG1 gene, IFFO1 gene in the preparation of a kit for in vitro detection of ovarian cancer.

[0277] The TFAP2E gene, as a transcription factor, can bind to specific sequences of DNA and regulate the expression of other genes. This regulatory effect plays a key role in cell proliferation, differentiation, migration and other processes.

[0278] The WNT6 gene, as a member of the Wnt signaling pathway, plays a key role in embryonic development, cell proliferation, differentiation, migration and tissue homeostasis. Specifically, it is involved in the regulation of various cellular processes, including stem cell maintenance, bone formation, tooth development, etc.

[0279] The EMX2OS gene, as a LncRNA, may function by regulating the expression of its adjacent coding gene EMX2. Studies have shown that LncRNA can be involved in a variety of biological processes, including transcriptional regulation, translational regulation, epigenetic regulation, etc.

[0280] The CRYBG1 gene plays an important role in cells, and the encoded protein can be involved in various biological processes. According to the disclosure of gene decoding technology, the places where the gene plays a role in cells include cytosol, microtubules, and nucleus. However, the specific function of the CRYBG1 gene and its mechanism of action in human diseases are still under study.

[0281] The IFFO1 gene encodes Intermediate Filament Family Orphan 1, which is a protein that may be involved in the maintenance of cytoskeletal structure and function in cells. Intermediate filaments are an important component of the cytoskeleton, and they play an important role in maintaining cell morphology, cell division, signal transduction, and cell response to external stimuli.

[0282] In another aspect, the present application provides a method for detecting ovarian cancer in vitro, comprising the following steps:

[0283] 1) isolating the target sequence or fragment of the target gene in the biological sample to be tested;

[0284] 2) determining the methylation state of the target sequence of the target gene;

[0285] 3) determining the state of the biological sample by the detection result of the methylation state of the target sequence of the target gene, thereby realizing the in vitro detection of ovarian cancer.

[0286] According to some preferred embodiments, the method further comprises the following steps:

[0287] 1) extracting genomic DNA from the biological sample to be tested;

[0288] 2) treating the DNA sample obtained in step 1) with a reagent to convert the 5-unmethylated cytosine bases to uracil or other bases, i.e. the 5-unmethylated cytosine bases of the target sequence of the target gene are converted to uracil or other bases, and the converted bases are different from the 5-unmethylated cytosine bases in terms of hybridization performance and are detectable;

[0289] 3) contacting the DNA sample treated in step 2) with a DNA polymerase and primers of the target sequence of the target gene, so that the treated target sequence of the target gene is amplified to produce an amplification product or is not amplified; the treated target sequence of the target gene will produce an amplification product if DNA polymerization reaction occurs; the treated target sequence of the target gene is not amplified if DNA polymerization reaction does not occur;

[0290] 4) detecting the amplification product with a probe; and

[0291] 5) determining the methylation state of at least one CpG dinucleotide of the target sequence of the gene of interest based on the presence or absence of the amplified product.

[0292] Preferably, the typical primer comprises a fragment of the target sequence of the gene of interest comprising a fragment of at least 9 nucleotides respectively identical, complementary or hybridizing under intermediate stringent or stringent conditions to a fragment selected from any one of SEQ ID NOs: 1-12, any one of SEQ ID NOs: 13-24, any one of SEQ ID NOs: 25-36, any one of SEQ ID NOs: 37-48, any one of SEQ ID NOs: 49-84.

[0293] Preferably, the typical probe comprises a fragment of the target sequence of the gene of interest comprising a fragment of at least 15 nucleotides respectively identical, complementary or hybridizing under intermediate stringent or stringent conditions to a fragment selected from any one of SEQ ID NOs: 1-12, any one of SEQ ID NOs: 13-24, any one of SEQ ID NOs: 25-36, any one of SEQ ID NOs: 37-48, any one of SEQ ID NOs: 49-84.

[0294] Preferably, one or more of the primers, probes are as shown in Table 2 above.

[0295] And, the contacting or amplifying comprises using at least one of the following methods: using a thermostable DNA polymerase as the amplification enzyme, using a polymerase lacking 5'-3' exonuclease activity, using polymerase chain reaction (PCR), generating amplified product nucleic acid molecules with a detectable label.

[0296] Preferably, the methylation status is determined by PCR, such as "fluorescence based real-time PCR technology", methylation sensitive single nucleotide primer extension reaction (Ms-SNuPE), methylation specific PCR (MSP), and methylation CpG island amplification (MCA) and the like. Among them, the "fluorescence based real-time PCR" assay is a high throughput quantitative methylation assay that uses fluorescence based real-time PCR (TaqMan) technology without further manipulation after the PCR step. Briefly, the "fluorescence based real-time PCR" method starts with a mixed sample of genomic DNA that is converted into a mixed pool of methylation dependent sequence differences in a sodium bisulfite reaction according to standard procedures. Subsequently, a fluorescence based PCR is performed in a "biased" reaction with PCR primers overlapping the known CpG dinucleotides. Sequence differences can be generated at the level of amplification as well as at the level of fluorescence detection of the amplification. The "fluorescence based real-time PCR" assay can be used as a quantitative test of the methylation status in a sample of genomic DNA, where sequence discrimination occurs at the level of probe hybridization. In this quantitative approach, the PCR reaction provides methylation specific amplification in the presence of a fluorescence probe overlapping the specific CpG dinucleotides. An unbiased control for the amount of starting DNA is provided by a reaction where neither primer nor probe covers any CpG dinucleotides. The "fluorescence based real-time PCR" method can be used with any suitable probe, such as "TaqMan", "Lightcycler" and the like. TaqMan probes are dual labeled with a fluorescent reporter (RTSPYL5rter) and a quencher molecule (Quencher) and are designed to be specific to a relatively high GC content region so that they melt at a temperature about 10°C higher than the forward or reverse primer in the PCR cycle. This allows the TaqMan probe to remain fully hybridized during the PCR annealing / extension step. As the Taq polymerase enzymatically synthesizes a new strand in the PCR, it eventually encounters the annealed TaqMan probe. The 5' to 3' exonuclease activity of the Taq polymerase then displaces it by digesting the TaqMan probe, thereby releasing the fluorescent reporter molecule for quantitative detection of its now unquenched signal using a real-time fluorescence detection system. Typical reagents for "fluorescence based real-time PCR" analysis can include, but are not limited to: PCR primers for the target sequence of the gene of interest; a non-specific amplification blocker; TaqMan or Lightcycler probes; optimized PCR buffer and deoxynucleotides; and Taq polymerase and the like.

[0297] In some preferred embodiments, the methylation state of at least one CpG dinucleotide in the target sequence of the target gene is determined by a critical Ct value of a real-time PCR reaction. By using the method for analyzing DNA in a biological sample by a real-time PCR reaction, the detection of the methylation state of the target sequence of the target gene can be easily achieved, and the critical Ct value of the PCR reaction can be used to quickly and conveniently determine whether the sample is positive, thus providing a non-invasive and rapid in vitro detection method for ovarian cancer.

[0298] The biological sample is selected from the group consisting of a cell line, a histological section, a tissue biopsy / paraffin-embedded tissue, a body fluid, feces, colon effluent, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, or a combination thereof. Preferably, the biological sample is plasma.

[0299] The present inventors have found that there is a significant difference in the methylation state of the target sequence of the TFAP2E gene, the WNT6 gene, the EMX2OS gene, the CRYBG1 gene, and the IFFO1 gene in ovarian cancer tissue and the methylation state of the target sequence of the gene in normal tissue: in ovarian cancer tissue, the target sequence of the TFAP2E gene, the WNT6 gene, the EMX2OS gene, the CRYBG1 gene, and the IFFO1 gene is methylated, while in normal tissue, the target sequence of the TFAP2E gene, the WNT6 gene, the EMX2OS gene, the CRYBG1 gene, and the IFFO1 gene is not methylated. Therefore, the present application provides a method for in vitro detection of ovarian cancer by detecting the methylation state of the target sequence of one or more of the TFAP2E gene, the WNT6 gene, the EMX2OS gene, the CRYBG1 gene, and the IFFO1 gene in a sample, and the method provided by the present application can non-invasively and rapidly detect ovarian cancer.

[0300] EMBODIMENT

[0301] The materials used in the experiments and the experimental methods are generally and / or specifically described in the present application, and in the following examples, % means wt%, i.e. weight percentage. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0302] Example 1 primer and probe test

[0303] Firstly, low methylation level sites were screened in the second-generation sequencing information of 340 healthy human white blood cell (WBC) and 102 healthy human plasma cfDNA samples. Secondly, the screened low methylation level sites were analyzed in plasma and ovarian cancer tissue, and the sites that were different in 103 ovarian cancer plasma and 102 normal human plasma and simultaneously different in 56 ovarian cancer tissues and 56 cancer-adjacent tissues were screened as candidate markers. Then, the candidate markers were traced back to plasma. Finally, the sensitivity and specificity of ovarian cancer tissue and normal human plasma were verified, and five specific markers of TFAP2E gene, WNT6 gene, EMX2OS gene, CRYBG1 gene and IFFO1 gene were determined. The primer and probe sequences were designed according to the target sequences of the above five genes, and the primer and probe sequences designed are shown in Table 2 above.

[0304] The DNA of the normal human white blood cell line is usually in a low / non-methylation state and can be used as a negative control. The amount of DNA used in this embodiment is 15.75 ng / reaction; the fully methylated DNA is in a high / fully methylated state and can be used as a positive control. The amount of DNA used in this embodiment is 200 pg / reaction. The DNA sample is first subjected to bisulfite conversion, and the converted BisDNA is used as a template for real-time PCR amplification using the above-mentioned primers and probes. The beta-actin (ACTB) gene is used as an internal reference, and the beta-actin gene amplicon is created by using primers complementary to the beta-actin gene sequence, and the beta-actin gene amplicon is detected with a specific probe. Each sample is subjected to at least one real-time PCR, and in some specific embodiments, two or three real-time PCR detections. The PCR system for primer and probe testing is shown in Table 4 below.

[0305] Table 4 PCR system for primer and probe testing Note: "F" indicates a forward primer; "R" indicates a reverse primer; "P" indicates a probe.

[0306] The PCR amplification program used is: 94°C, 20 min; (93°C, 30 s; 57°C, 35 s-read fluorescence signal) 45 cycles; 40°C, 5 s.

[0307] The results are shown in Table 5 below. When the BisDNA of the fully methylated DNA is used as a template, the TFAP2E gene, WNT6 gene, EMX2OS gene, CRYBG1 gene and IFFO1 gene can be effectively amplified; and when the BisDNA of the WBC is used as a template, the target genes other than the internal reference gene ACTB are not amplified.

[0308] Table 5 Results of primer and probe testing of each gene Note: "No Ct" means that the Ct value is not detected.

[0309] Example 2

[0310] Sixteen samples of ovarian cancer tissue (10 ng / reaction) and sixteen samples of normal human plasma (3.5 mL) were selected, genomic DNA was extracted, and after bisulfite conversion, the methylation of the TFAP2E gene, the WNT6 gene, the EMX2OS gene, the CRYBG1 gene, and the IFFOl gene was detected according to the PCR reaction systems in Tables 6-10 and the reaction procedures in Example 1. Finally, the Ct values of the real-time PCR of the target sequences of the genes in the sixteen samples of ovarian cancer tissue and the sixteen samples of normal human plasma were measured, and the results are shown in Tables 11-12.

[0311] Table 6 PCR reaction system of TFAP2E

[0312] Table 7 PCR reaction system of WNT6

[0313] Table 8 PCR reaction system of EMX2OS

[0314] Table 9 PCR reaction system of CRYBG1

[0315] Table 10 PCR reaction system of IFFOl Note: In Tables 6-10, "F" represents the forward primer; "R" represents the reverse primer; and "P" represents the probe.

[0316] Table 11 Sensitivity detection of each gene in ovarian cancer samples

[0317] Table 12 Specificity detection of each gene in benign samples

[0318] The results of Tables 11 and 12 show that the detection of ovarian cancer tissue and benign samples using the TFAP2E gene, the WNT6 gene, the EMX2OS gene, the CRYBG1 gene, and the IFFOl gene can achieve good sensitivity and specificity. When interpreting, first, according to the amplification of the gene, it is determined whether the detection result of the gene is positive (when the Ct value of the marker is less than 41, it is determined that the sample is positive in the detection result of the gene); and then, according to the gene result, it is determined whether the sample under test is positive.

[0319] For the TFAP2E gene, the WNT6 gene and the IFF01 gene, one sequence with the best sensitivity and specificity was selected, i.e. TFAP2E-1, WNT6-2 and IFF01-1, respectively. The sensitivity of the detection of the three genes alone was 87.50%, 81.25% and 87.50%, respectively. The sensitivity of the final judgment reached 100% when any two of the three genes were detected positively. Meanwhile, the methylation of the target sequence of the objective gene had good specificity. The specificity of TFAP2E-1 was 93.75%, the specificity of WNT6-2 was 93.75%, the specificity of IFF01-1 was 100%, and the specificity of the three genes being negative was 93.75%.

[0320] For the EMX2OS gene, the CRYBG1 gene and the IFF01 gene, one sequence with the best sensitivity and specificity was selected, i.e. EMX2OS-1, CRYBG1-2 and IFF01-4, respectively. The sensitivity of the detection of the three genes alone was 87.50%, 87.50% and 81.25%, respectively. The sensitivity of the final judgment reached 100% when any two of the three genes were detected positively. Meanwhile, the methylation of the target sequence of the objective gene had good specificity. The specificity of EMX2OS and IFF01 both reached 93.75%, the specificity of CRYBG1 was 100%, and the specificity of the three genes being negative was 93.75%.

[0321] For the IFF01 gene, the sensitivity of IFF01-7 alone in the detection of ovarian cancer tissues was 100%, and the corresponding specificity reached 93.75%.

[0322] In the above-mentioned five markers related to ovarian cancer, the tissue sensitivity of the five markers was above 81.25% from the detection results of tissues, and the sensitivity of the combined judgment of the three genes reached 100%. From the detection results of the plasma of healthy people, the plasma specificity of the single gene was above 90.75%, and the combined judgment of the three genes reached 93.75%. Under the condition of small sample quantity verification, the plasma specificity of 80% was a relatively reasonable and high index level. Therefore, the above experimental results showed that the five objective genes selected in the application were potential markers for the detection of the methylation of ovarian cancer.

[0323] Example 3

[0324] Thirty-seven plasma samples (3.5 mL) of ovarian cancer (samples S1-S37) and thirty-three plasma samples (3.5 mL) of normal people (samples S38-S70) were selected, genomic DNA was extracted, and after bisulfite conversion, the PCR reaction system in Example 1 was used for methylation detection in combination with TFAP2E-1, WNT6-2 and IFF01-1 genes. Finally, the Ct values of real-time PCR of the target sequences of the genes in the 37 plasma samples of ovarian cancer and the 33 plasma samples of normal people were measured (when the Ct value was less than 41, the sample was determined to be positive in the gene detection result), and the result was determined to be positive when two of the three genes were detected to be positive, and the rest was determined to be negative. The results are shown in Tables 13-15. The sensitivity of TFAP2E-1, WNT6-2 and IFF01-1 genes for detecting ovarian cancer alone was 75.68%, 78.39% and 73.00%, respectively; the sensitivity of the three genes in combination reached 91.89%, and the specificity was 93.94%.

[0325] Table 13 Sensitivity of each gene in the plasma sample of ovarian cancer

[0326] Table 14 Specificity of each gene in the plasma sample of normal people

[0327] Table 15

[0328] The above experimental results show that the methylation DNA of the target sequence of the gene is a marker of ovarian cancer. Through the detection of the methylation DNA of the target sequence of the gene in the present application, in vitro non-invasive detection of ovarian cancer can be achieved, and the detection rate of ovarian cancer can be improved.

[0329] Example 4

[0330] Thirty-five plasma samples (3.5 mL) of ovarian cancer and thirty-one plasma samples (3.5 mL) of normal persons were collected, and genomic DNA was extracted. After the genomic DNA was converted into BisDNA by bisulfite, the PCR reaction system in Example 1 was used, and the methylation of the target sequence of the gene was detected. Finally, the Ct value of the real-time PCR of the target sequence of the gene in the thirty-five plasma samples of ovarian cancer and the thirty-one plasma samples of normal persons was measured (when the Ct value was less than 41, the sample was determined to be positive in the gene detection result), and the result was determined to be negative according to the positive detection of two of the three genes. The results are shown in Tables 16-17. The sensitivity of the EMX2OS-1, CRYBG1-2 and IFFO1-4 genes for detecting ovarian cancer was 77.14%, 74.29% and 77.14%, respectively. The sensitivity of any two or three genes of the three genes was 91.43%, and the specificity was 93.55%.

[0331] Table 16 Sensitivity of each gene in the plasma sample of ovarian cancer

[0332] Table 17 Specificity of each gene in the plasma sample of normal persons

[0333] The above experimental results show that the methylation DNA of the target sequence of the gene is a marker of ovarian cancer. Through the detection of the methylation DNA of the target sequence of the gene, the in vitro non-invasive detection of ovarian cancer can be realized, and the detection rate of ovarian cancer can be improved.

[0334] Example 5

[0335] Thirty-eight plasma samples of benign tumors, thirty-four plasma samples of normal persons and forty-two plasma samples of ovarian cancer were collected from a hospital, and genomic DNA was extracted. After the genomic DNA was converted into BisDNA by bisulfite, the PCR reaction system in Example 1 was used, and the methylation of the target sequence of the gene was detected. Finally, the negative result was determined according to the obtained ct value, and the ct value ≥ 41 was used as the cut-off value.

[0336] The reaction system was used to detect 114 samples, and the detection results are shown in Table 18. The sensitivity of the IFFO1 gene for detecting ovarian cancer was 90.5%, the specificity for detecting benign plasma samples was 73.7%, and the specificity for detecting normal plasma samples was 91.2%. The performance indicators are shown in Table 19.

[0337] Table 18 Detection results of IFFO1 gene in cancer plasma and normal plasma

[0338] Table 19 Performance index of IFFO1 gene

[0339] The above experimental results show that the methylation DNA of the target sequence of the target gene is a marker of ovarian cancer. Through the detection of the methylation DNA of the target sequence of the target gene, the in vitro non-invasive detection of ovarian cancer can be realized, and the detection rate of ovarian cancer can be improved.

[0340] In summary, by using the above-mentioned composition, nucleic acid sequence, kit and use thereof, and the above-mentioned detection method, by detecting the methylation of the nucleic acid sequence of the target sequence of the target gene and its fragment, the in vitro detection of ovarian cancer by using the methylation biomarker of the target sequence of the target gene is realized, thereby effectively improving the sensitivity and specificity of the in vitro detection of ovarian cancer.

[0341] The above-mentioned is only the preferred embodiment of the present application, and does not limit the present application in other forms. Any person skilled in the art can modify or change the above-mentioned disclosed technical content into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change and modification made on the above-mentioned embodiments without departing from the technical content of the present application, according to the technical essence of the present application, still belongs to the protection scope of the technical solution of the present application.

Claims

1. A composition for detecting ovarian cancer in vitro, the composition comprising: a nucleic acid for detecting a methylation state of a target gene, wherein the methylation state of the target gene is characterized by methylation of a target sequence of the target gene, and wherein the target gene is one or two or more of a TFAP2E gene, a WNT6 gene, an EMX2OS gene, a CRYBG1 gene, and an IFFO1 gene. The target gene is the TFAP2E gene, the WNT6 gene, and the IFFO1 gene; or the EMX2OS gene, the CRYBG1 gene, and the IFFO1 gene. The target sequence of the TFAP2E gene is as shown in any one of SEQ ID NOs: 1-12 or comprises a sequence as shown in any one of SEQ ID NOs: 1-12. Preferably, the target sequence of the TFAP2E gene comprises a sequence as shown in any one of SEQ ID NOs: 1-4 and any one of SEQ ID NOs: 5-8, or a sequence as shown in any one of SEQ ID NOs: 1-4 and any one of SEQ ID NOs: 9-12, or a sequence as shown in any one of SEQ ID NOs: 5-8 and any one of SEQ ID NOs: 9-12, or a sequence as shown in any one of SEQ ID NOs: 1-4 and any one of SEQ ID NOs: 5-8 and any one of SEQ ID NOs: 9-12.

2. The composition of claim 1, wherein, The target sequence of the WNT6 gene is as shown in any one of SEQ ID NOs: 13-24 or comprises a sequence as shown in any one of SEQ ID NOs: 13-24.

3. The composition of claim 1, wherein, Preferably, the target sequence of the WNT6 gene comprises a sequence as shown in any one of SEQ ID NOs: 13-16 and any one of SEQ ID NOs: 17-20, or a sequence as shown in any one of SEQ ID NOs: 13-16 and any one of SEQ ID NOs: 21-24, or a sequence as shown in any one of SEQ ID NOs: 17-20 and any one of SEQ ID NOs: 21-24, or a sequence as shown in any one of SEQ ID NOs: 13-16 and any one of SEQ ID NOs: 17-20 and any one of SEQ ID NOs: 21-24. The target sequence of the EMX2OS gene is as shown in any one of SEQ ID NOs: 25-36 or comprises a sequence as shown in any one of SEQ ID NOs: 25-36.

4. The composition of claim 1, wherein, Preferably, the target sequence of the EMX2OS gene comprises a sequence as shown in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 29-32, or a sequence as shown in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 33-36, or a sequence as shown in any one of SEQ ID NOs: 29-32 and any one of SEQ ID NOs: 33-36, or a sequence as shown in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 29-32 and any one of SEQ ID NOs: 33-36. ​ 5. The composition of claim 1, wherein, ​ Preferably, the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 29-32, or the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 33-36, or the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 29-32 and any one of SEQ ID NOs: 33-36, or the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 29-32 and any one of SEQ ID NOs: 33-36.

6. The composition of claim 1, wherein, the target sequence of the CRYBG1 gene is as set forth in any one of SEQ ID NOs: 37-48 or the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 37-48; Preferably, the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 37-40 and any one of SEQ ID NOs: 41-44, or the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 37-40 and any one of SEQ ID NOs: 45-48, or the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 41-44 and any one of SEQ ID NOs: 45-48, or the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 37-40 and any one of SEQ ID NOs: 41-44 and any one of SEQ ID NOs: 45-48.

7. The composition of claim 1, wherein, the target sequence of the IFF01 gene is as set forth in any one of SEQ ID NOs: 49-84 or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-84; Preferably, the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 53-56, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 57-60, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 53-56 and any one of SEQ ID NOs: 57-60, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 53-56 and any one of SEQ ID NOs: 57-60. Preferably, the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 53-56, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 57-60, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 53-56 and any one of SEQ ID NOs: 57-60, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 53-56 and any one of SEQ ID NOs: 57-60, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 61-64 and any one of SEQ ID NOs: 65-68, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 61-64 and any one of SEQ ID NOs: 69-72, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 65-68 and any one of SEQ ID NOs: 69-72, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 61-64 and any one of SEQ ID NOs: 65-68 and any one of SEQ ID NOs: 69-72, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 73-76 and any one of SEQ ID NOs: 77-80, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 73-76 and any one of SEQ ID NOs: 81-84, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 77-80 and any one of SEQ ID NOs: 81-84, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 73-76 and any one of SEQ ID NOs: 77-80 and any one of SEQ ID NOs: 81-84.

8. The composition according to any one of claims 1-7, wherein, The nucleic acid for detecting the methylation state of the target gene comprises: a primer which is a fragment of at least 9 nucleotides in the target sequence of the target gene, the fragment comprises at least one CpG dinucleotide sequence; Preferably, the fragment of at least 9 nucleotides is a sequence as set forth in SEQ ID NO: 85 and SEQ ID NO: 86, a sequence as set forth in SEQ ID NO: 88 and SEQ ID NO: 89, a sequence as set forth in SEQ ID NO: 91 and SEQ ID NO: 92, a sequence as set forth in SEQ ID NO: 94 and SEQ ID NO: 95, a sequence as set forth in SEQ ID NO: 97 and SEQ ID NO: 98, a sequence as set forth in SEQ ID NO: 100 and SEQ ID NO: 101, a sequence as set forth in SEQ ID NO: 103 and SEQ ID NO: 104, a sequence as set forth in SEQ ID NO: 106 and SEQ ID NO: 107, a sequence as set forth in SEQ ID NO: 109 and SEQ ID NO: 110, a sequence as set forth in SEQ ID NO: 112 and SEQ ID NO: 113, a sequence as set forth in SEQ ID NO: 115 and SEQ ID NO: 116, a sequence as set forth in SEQ ID NO: 118 and SEQ ID NO: 119, a sequence as set forth in SEQ ID NO: 121 and SEQ ID NO: 122, a sequence as set forth in SEQ ID NO: 124 and SEQ ID NO: 125, a sequence as set forth in SEQ ID NO: 127 and SEQ ID NO: 128, a sequence as set forth in SEQ ID NO: 130 and SEQ ID NO: 131, a sequence as set forth in SEQ ID NO: 133 and SEQ ID NO: 134, a sequence as set forth in SEQ ID NO: 136 and SEQ ID NO: 137, a sequence as set forth in SEQ ID NO: 139 and SEQ ID NO: 140, a sequence as set forth in SEQ ID NO: 142 and SEQ ID NO: 143, or a sequence as set forth in SEQ ID NO: 145 and SEQ ID NO:

146.

9. The composition according to any one of claims 1-8, wherein, The nucleic acid for detecting the methylation state of the target gene comprises: a probe which is a fragment of at least 15 nucleotides hybridizing to a target sequence of the target gene under medium stringency or high stringency conditions, the fragment comprising at least one CpG dinucleotide sequence; Preferably, the fragment of at least 15 nucleotides is the sequence of SEQ ID NO: 87, the sequence of SEQ ID NO: 90, the sequence of SEQ ID NO: 93, the sequence of SEQ ID NO: 96, the sequence of SEQ ID NO: 99, the sequence of SEQ ID NO: 102, the sequence of SEQ ID NO: 105, the sequence of SEQ ID NO: 108, the sequence of SEQ ID NO: 111, the sequence of SEQ ID NO: 114, the sequence of SEQ ID NO: 117, the sequence of SEQ ID NO: 120, the sequence of SEQ ID NO: 123, the sequence of SEQ ID NO: 126, the sequence of SEQ ID NO: 129, the sequence of SEQ ID NO: 132, the sequence of SEQ ID NO: 135, the sequence of SEQ ID NO: 138, the sequence of SEQ ID NO: 141, the sequence of SEQ ID NO: 144, or the sequence of SEQ ID NO:

147.

10. The composition according to any one of claims 1-9, wherein, The composition further comprises: An agent that converts an unmethylated cytosine base at position 5 of a target sequence of a target gene into uracil.

11. The composition of any one of claims 1-10, wherein the nucleic acid for detecting the methylation status of a target gene further comprises: A blocker that preferentially binds to a target sequence in an unmethylated state.

12. An oligonucleotide for detecting ovarian cancer in vitro, comprising: a fragment of at least 9 nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1-12 or a complement thereof and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 9 nucleotides of a sequence as set forth in any one of SEQ ID NOs: 13-24 or a complement thereof and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 9 nucleotides of a sequence as set forth in any one of SEQ ID NOs: 25-36 or a complement thereof and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 9 nucleotides of a sequence as set forth in any one of SEQ ID NOs: 37-48 or a complement thereof and comprising at least one CpG dinucleotide sequence; and / or a fragment of at least 9 nucleotides of a sequence as set forth in any one of SEQ ID NOs: 49-84 or a complement thereof and comprising at least one CpG dinucleotide sequence.

13. The oligonucleotide of claim 12, further comprising: a fragment of at least 15 nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1-12 or a complement thereof and comprising at least one CpG dinucleotide sequence that hybridizes under medium stringency or high stringency conditions; and / or a fragment of at least 15 nucleotides of a sequence as set forth in any one of SEQ ID NOs: 13-24 or a complement thereof and comprising at least one CpG dinucleotide sequence that hybridizes under medium stringency or high stringency conditions; and / or a fragment of at least 15 nucleotides of a sequence as set forth in any one of SEQ ID NOs: 25-36 or a complement thereof and comprising at least one CpG dinucleotide sequence that hybridizes under medium stringency or high stringency conditions; and / or a fragment of at least 15 nucleotides of a sequence as set forth in any one of SEQ ID NOs: 37-48 or a complement thereof and comprising at least one CpG dinucleotide sequence that hybridizes under medium stringency or high stringency conditions; and / or a fragment of at least 15 nucleotides of a sequence as set forth in any one of SEQ ID NOs: 49-84 or a complement thereof and comprising at least one CpG dinucleotide sequence that hybridizes under medium stringency or high stringency conditions. Fragments that hybridize to at least 15 nucleotides of the sequences shown in any one of SEQ ID NO:25-36 or their complementary sequences under moderately or strictly controlled conditions, and containing at least one CpG dinucleotide sequence; and / or Fragments that hybridize to at least 15 nucleotides of the sequences shown in any one of SEQ ID NO:37-48 or their complementary sequences under moderately or strictly controlled conditions, and containing at least one CpG dinucleotide sequence; and / or A fragment that hybridizes to at least 15 nucleotides of the sequence shown in any one of SEQ ID NO:49-84 or its complementary sequence under moderately or strictly controlled conditions and contains at least one CpG dinucleotide sequence.

14. The oligonucleotide of claim 12, further comprising: Blockers that preferentially bind to target sequences in an unmethylated state.

15. An oligonucleotide for in vitro detection of ovarian cancer, comprising: The sequences of SEQ ID NO:85 and SEQ ID NO:86, preferably, also include the sequence of SEQ ID NO:87; or The sequences of SEQ ID NO:88 and SEQ ID NO:89, preferably, also include the sequence of SEQ ID NO:90; or The sequences of SEQ ID NO:91 and SEQ ID NO:92, preferably, also include the sequence of SEQ ID NO:93; or The sequences of SEQ ID NO:94 and SEQ ID NO:95, preferably, also include the sequence of SEQ ID NO:96; or The sequences of SEQ ID NO:97 and SEQ ID NO:98, preferably, also include the sequence of SEQ ID NO:99; or The sequences of SEQ ID NO:100 and SEQ ID NO:101, preferably also including the sequence of SEQ ID NO:102; or The sequences of SEQ ID NO:103 and SEQ ID NO:104, preferably, also include the sequence of SEQ ID NO:105; or The sequences of SEQ ID NO:106 and SEQ ID NO:107, preferably, also include the sequence of SEQ ID NO:108; or The sequences of SEQ ID NO:109 and SEQ ID NO:110, preferably, also include the sequence of SEQ ID NO:111; or The sequences of SEQ ID NO:112 and SEQ ID NO:113, preferably, also include the sequence of SEQ ID NO:114; or The sequences of SEQ ID NO:115 and SEQ ID NO:116, preferably, also include the sequence of SEQ ID NO:117; or The sequences of SEQ ID NO:118 and SEQ ID NO:119, preferably, also include the sequence of SEQ ID NO:120; or the sequence of SEQ ID NO: 121 and the sequence of SEQ ID NO: 122, preferably further comprising the sequence of SEQ ID NO: 123; or the sequence of SEQ ID NO: 124 and the sequence of SEQ ID NO: 125, preferably further comprising the sequence of SEQ ID NO: 126; or the sequence of SEQ ID NO: 127 and the sequence of SEQ ID NO: 128, preferably further comprising the sequence of SEQ ID NO: 129; or the sequence of SEQ ID NO: 130 and the sequence of SEQ ID NO: 131, preferably further comprising the sequence of SEQ ID NO: 132; or the sequence of SEQ ID NO: 133 and the sequence of SEQ ID NO: 134, preferably further comprising the sequence of SEQ ID NO: 135; or the sequence of SEQ ID NO: 136 and the sequence of SEQ ID NO: 137, preferably further comprising the sequence of SEQ ID NO: 138; or the sequence of SEQ ID NO: 139 and the sequence of SEQ ID NO: 140, preferably further comprising the sequence of SEQ ID NO: 141; or the sequence of SEQ ID NO: 142 and the sequence of SEQ ID NO: 143, preferably further comprising the sequence of SEQ ID NO: 144; or the sequence of SEQ ID NO: 145 and the sequence of SEQ ID NO: 146, preferably further comprising the sequence of SEQ ID NO:

147.

16. A kit comprising the composition of any one of claims 1-11 or comprising the oligonucleotide of any one of claims 12-15.

17. The kit of claim 16, further comprising at least one additional component selected from the group consisting of: nucleotides triphosphates, a DNA polymerase, and a buffer required for the function of said DNA polymerase.

18. The kit of claim 16 or 17, wherein, the sample for detection of said kit comprises: a cell line, a histological section, a tissue biopsy / paraffin-embedded tissue, a body fluid, a stool, a colonic effluent, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, or a combination thereof.

19. The kit of any one of claims 16-18, further comprising: an instruction.

20. Use of the composition of any one of claims 1-11 or the oligonucleotide of any one of claims 12-15 in the manufacture of a kit for in vitro detection of ovarian cancer.

21. The use according to claim 20, wherein, said kit for in vitro detection of ovarian cancer detects ovarian cancer by a method comprising the following steps: 1) isolating a DNA sample comprising a target sequence of a target gene or a fragment thereof in a biological sample to be tested; 2) determining the methylation status of the target sequence of the target gene; 3) judging the state of the biological sample by the detection result of the methylation status of the target sequence of the target gene, thereby achieving in vitro detection of ovarian cancer.

22. The use of claim 21, wherein, said method comprises the following steps: extracting genomic DNA of a biological sample to be tested; treating the extracted genomic DNA with a reagent to convert 5 unmethylated cytosine bases to uracil or other bases; contacting the reagent-treated DNA sample with a DNA polymerase and primers for a target sequence of the target gene, and performing a DNA polymerization reaction; detecting the amplification product with a probe; and determining the methylation state of at least one CpG dinucleotide of the target sequence of the target gene based on whether the amplification product is present or not.

23. The use of claim 22, wherein, The reagent is a bisulfite reagent.

24. Use of one or more of a TFAP2E gene, a WNT6 gene, an EMX2OS gene, a CRYBG1 gene, and an IFFO1 gene in the preparation of a kit for detecting ovarian cancer in vitro.

25. The use of claim 24, wherein, The target sequence of the TFAP2E gene is as set forth in any one of SEQ ID NOs: 1-12 or the target sequence of the TFAP2E gene comprises a sequence as set forth in any one of SEQ ID NOs: 1-12; Preferably, the target sequence of the TFAP2E gene comprises a sequence as set forth in any one of SEQ ID NOs: 1-4 and any one of SEQ ID NOs: 5-8, or the target sequence of the TFAP2E gene comprises a sequence as set forth in any one of SEQ ID NOs: 1-4 and any one of SEQ ID NOs: 9-12, or the target sequence of the TFAP2E gene comprises a sequence as set forth in any one of SEQ ID NOs: 5-8 and any one of SEQ ID NOs: 9-12, or the target sequence of the TFAP2E gene comprises a sequence as set forth in any one of SEQ ID NOs: 1-4 and any one of SEQ ID NOs: 5-8 and any one of SEQ ID NOs: 9-12.

26. The use of claim 24, wherein, The target sequence of the WNT6 gene is as set forth in any one of SEQ ID NOs: 13-24 or the target sequence of the WNT6 gene comprises a sequence as set forth in any one of SEQ ID NOs: 13-24; Preferably, the target sequence of the WNT6 gene comprises a sequence as set forth in any one of SEQ ID NOs: 13-16 and any one of SEQ ID NOs: 17-20, or the target sequence of the WNT6 gene comprises a sequence as set forth in any one of SEQ ID NOs: 13-16 and any one of SEQ ID NOs: 21-24, or the target sequence of the WNT6 gene comprises a sequence as set forth in any one of SEQ ID NOs: 17-20 and any one of SEQ ID NOs: 21-24, or the target sequence of the WNT6 gene comprises a sequence as set forth in any one of SEQ ID NOs: 13-16 and any one of SEQ ID NOs: 17-20 and any one of SEQ ID NOs: 21-24.

27. The use of claim 24, wherein, The target sequence of the EMX2OS gene is as set forth in any one of SEQ ID NOs: 25-36 or the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 25-36; Preferably, the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 29-32, or the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 33-36, or the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 29-32 and any one of SEQ ID NOs: 33-36, or the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 29-32 and any one of SEQ ID NOs: 33-36. Preferably, the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 29-32, or the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 33-36, or the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 29-32 and any one of SEQ ID NOs: 33-36, or the target sequence of the EMX2OS gene comprises a sequence as set forth in any one of SEQ ID NOs: 25-28 and any one of SEQ ID NOs: 29-32 and any one of SEQ ID NOs: 33-36.

28. The use of claim 24, wherein, the target sequence of the CRYBG1 gene is as set forth in any one of SEQ ID NOs: 37-48 or the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 37-48; Preferably, the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 37-40 and any one of SEQ ID NOs: 41-44, or the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 37-40 and any one of SEQ ID NOs: 45-48, or the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 41-44 and any one of SEQ ID NOs: 45-48, or the target sequence of the CRYBG1 gene comprises a sequence as set forth in any one of SEQ ID NOs: 37-40 and any one of SEQ ID NOs: 41-44 and any one of SEQ ID NOs: 45-48.

29. The use of claim 24, wherein, the target sequence of the IFF01 gene is as set forth in any one of SEQ ID NOs: 49-84 or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-84; Preferably, the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 53-56, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 57-60, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 53-56 and any one of SEQ ID NOs: 57-60, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 53-56 and any one of SEQ ID NOs: 57-60. Preferably, the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 53-56, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 57-60, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 53-56 and any one of SEQ ID NOs: 57-60, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 49-52 and any one of SEQ ID NOs: 53-56 and any one of SEQ ID NOs: 57-60, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 61-64 and any one of SEQ ID NOs: 65-68, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 61-64 and any one of SEQ ID NOs: 69-72, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 65-68 and any one of SEQ ID NOs: 69-72, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 61-64 and any one of SEQ ID NOs: 65-68 and any one of SEQ ID NOs: 69-72, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 73-76 and any one of SEQ ID NOs: 77-80, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 73-76 and any one of SEQ ID NOs: 81-84, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 77-80 and any one of SEQ ID NOs: 81-84, or the target sequence of the IFF01 gene comprises a sequence as set forth in any one of SEQ ID NOs: 73-76 and any one of SEQ ID NOs: 77-80 and any one of SEQ ID NOs: 81-84.

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