CN118308481A

CN118308481A - Biomarker, primer pair, nucleic acid product, kit and application for diagnosing ovarian cancer

Info

Publication number: CN118308481A
Application number: CN202410306499.2A
Authority: CN
Inventors: 刘成杰; 刘严; 洪瑶; 周谛晗; 孙帆; 董兰兰
Original assignee: Wuhan Aimisen Life Technology Co ltd
Current assignee: Wuhan Aimisen Life Technology Co ltd
Priority date: 2024-03-18
Filing date: 2024-03-18
Publication date: 2024-07-09

Abstract

The application discloses a biomarker, a primer pair, a nucleic acid product, a kit and application for diagnosing ovarian cancer. The marker comprises the full length or partial region of the positive strand of Ch10: 93567151-93567551 of the GPR120 gene and/or the full length or partial region of the negative strand of Ch18: 3499055-3499455 of the DLGAP1 gene. By detecting the methylation level of a marker in a sample, ovarian cancer patients can be distinguished from healthy persons. The application also discloses products such as a primer pair, a probe, a kit and the like for detecting the marker, and the products can effectively detect the methylation level of the marker in a tissue or blood sample, so that the sensitivity and the specificity of early diagnosis are improved while the noninvasive detection of the ovarian cancer is realized.

Description

Biomarker, primer pair, nucleic acid product, kit and application for diagnosing ovarian cancer

Technical Field

The application relates to the technical field of ovarian cancer diagnosis, in particular to a biomarker, a primer pair, a nucleic acid product, a kit and application for diagnosing ovarian cancer.

Background

Ovarian cancer (Ovarian Cancer) is one of the most common gynecological malignant tumors, is easy to metastasize and widely spread, and has incidence rate which is the third place of the gynecological malignant tumors and is only lower than cervical cancer and endometrial cancer. The mortality rate of ovarian cancer is always high, the survival rate of the ovarian cancer in the last 5 years is the lowest in gynecological tumors and is only 46%, and 15% of patients die within two months of definite diagnosis, so that the survival rate of the ovarian cancer and the survival quality of the patients are imperative to be improved. Ovarian cancer can reach 92% of survival rates in the near 5 years if found early, but early diagnosis of ovarian cancer is a major problem.

The existing clinical screening and diagnosis methods of ovarian cancer can be classified into three main categories: imaging detection, such as transvaginal ultrasound, CT, PET/CT, MRI, etc.; serology, such as detection of tumor markers in serum, such as CA-125, and pathology detection, such as tissue biopsies, cytology assays, and the like. However, these methods have unavoidable drawbacks: the main defect of imaging detection is that the detection result is lagged, and biopsy is needed for confirming diagnosis later; the sensitivity and the specificity of serological marker detection are poor; pathology tests such as cytology tests and tissue biopsies are invasive, patient compliance in diagnosis and treatment is low, sensitivity and repeatability are poor, and pathology tests are too dependent on subjective judgment of a pathologist, etc.

Therefore, finding new noninvasive, highly accurate methods for early diagnosis and screening of ovarian cancer is currently a problem to be solved in clinical urgent need.

Disclosure of Invention

Aiming at the defects of the prior art, the application provides a biomarker, a primer pair, a nucleic acid product, a kit and application for diagnosing ovarian cancer, wherein the biomarker can effectively distinguish ovarian cancer patients from healthy people, and the primer and the kit provided by the application can accurately detect the level of the marker by using blood as a detection sample, so that the high-sensitivity, high-specificity and noninvasive early diagnosis of ovarian cancer is realized.

In order to achieve the above purpose, the present application provides the following technical solutions:

in a first aspect, the application provides a biomarker for diagnosing ovarian cancer comprising:

A first nucleic acid fragment comprising the full length or a partial region of the chr10:93567151-93567551 positive strand of the GPR120 gene;

And/or

A second nucleic acid fragment comprising the full length or a partial region of the negative strand of chr18:3499055-3499455 of the DLGAP gene.

Further, the nucleotide sequence of the first nucleic acid fragment is shown as SEQ ID NO. 1; the nucleotide sequence of the second nucleic acid fragment is shown as SEQ ID NO. 2.

In a second aspect, the present application provides a primer pair for detecting the aforementioned biomarker, comprising:

A first primer pair comprising a first methylated primer pair comprising a first upstream methylated primer and a first downstream methylated primer, the nucleotide sequences of the first upstream methylated primer and the first downstream methylated primer being shown in SEQ ID NO.7 and SEQ ID NO.8, respectively;

And/or

And a second primer pair comprising a second methylated primer pair comprising a second upstream methylated primer and a second downstream methylated primer, the nucleotide sequences of the second upstream methylated primer and the second downstream methylated primer being shown in SEQ ID NO.11 and SEQ ID NO.12, respectively.

Further, the first primer pair further comprises a first unmethylated primer pair, the first unmethylated primer pair comprises a first upstream unmethylated primer and a first downstream unmethylated primer, and the nucleotide sequences of the first upstream unmethylated primer and the first downstream unmethylated primer are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10; when the primer pair comprises the second primer pair, the second primer pair further comprises a second unmethylated primer pair, the second unmethylated primer pair comprises a second upstream unmethylated primer and a second downstream unmethylated primer, and the nucleotide sequences of the second upstream unmethylated primer and the second downstream unmethylated primer are respectively shown as SEQ ID NO.13 and SEQ ID NO. 14.

In a third aspect, the present application provides a probe set for detecting the aforementioned biomarker, comprising at least one of the probes having the nucleotide sequences shown in SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO. 19.

In a fourth aspect, the application provides a nucleic acid product for the detection of ovarian cancer comprising the aforementioned primer pair and/or the aforementioned probe set.

In a fifth aspect, the application provides a kit for the detection of ovarian cancer comprising the nucleic acid product as described above.

In a sixth aspect, the application provides the use of the aforementioned biomarker or the aforementioned primer pair or the aforementioned probe set or the aforementioned nucleic acid product or the aforementioned kit for the preparation of an ovarian cancer diagnostic product.

The application provides a biomarker, a primer pair, a nucleic acid product, a kit and application for diagnosing ovarian cancer, and compared with the prior art, the application has at least the following beneficial effects:

(1) According to the methylation level of the ovarian cancer marker provided by the application, an ovarian cancer patient and a healthy person can be effectively distinguished, and the ovarian cancer marker has higher diagnosis sensitivity and specificity to ovarian cancer.

(2) The sensitivity of detection products such as the detection kit provided by the application for detecting ovarian cancer can reach 86.5%, and the specificity of detection of healthy people can reach 92.8%.

(3) The marker, the primer and the kit provided by the application can be used for non-invasive detection, early screening and improvement of prognosis of patients of ovarian cancer, and have important significance.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.

Interpretation of the terms

In the present disclosure, the terms "first," "second," and the like are used to distinguish between identical items or similar items that have substantially the same function and action, but are merely used to clearly describe the technical solutions of the embodiments of the present disclosure, and are not to be construed as indicating or implying a relative importance or implying that the number of technical features indicated is indicated.

In the context of the present application, the term "diagnosis" includes use in the auxiliary diagnosis of ovarian cancer, risk of recurrence assessment, assessment of risk and extent of cancerous lesions, prognosis, and the like.

In the context of the present application, the term "DNA methylation" is a form of chemical modification of DNA that is capable of altering genetic manifestations without altering the DNA sequence, where DNA methylation refers to covalent bonding of a methyl group at the cytosine 5 carbon of a genomic CpG site under the action of a DNA methyltransferase, and where DNA methylation can cause alterations in chromatin structure, DNA conformation, DNA stability, and the manner in which DNA interacts with proteins, thereby controlling gene expression. In the context of the present application, terms such as "methylation" are to be understood as being equivalent to the definition of the term "DNA methylation". In the context of the present application, "fully methylated" means that all CpG sites in a region segment are methylated, "partially methylated" means that CpG sites in a region segment are partially unmethylated, and "fully unmethylated" means that all CpG sites in a region segment are unmethylated.

In the context of the present application, the term "CpG site" refers to a structure in which deoxycytosine (Cytosine, c) and deoxyguanine (Guanine, c) are linked by a Phosphate (Phosphate, p) bond in a deoxynucleotide sequence, cpG is generally present on a DNA molecule, and two adjacent bases are distributed more densely in some specific DNA regions (called "CpG islands"), and CpG islands are located in a gene promoter region and are closely related to transcriptional regulation of a gene.

In the context of the present application, the term "methylation state" refers to a state determined according to whether or not cytosine in one or more CpG sites of a DNA marker fragment is methylated or the frequency/proportion/percentage of methylation, and may include methylation positive and methylation negative states, representing a qualitative concept. For example, if the frequency/proportion/percentage of methylation of cytosines in a CpG site in a DNA marker fragment exceeds a certain threshold, the methylation status of the DNA marker fragment may be determined to be methylation positive, otherwise the methylation status of the DNA marker fragment may be determined to be methylation negative. In practical application, different methylation states of the DNA marker fragments can be detected according to practical situations, for example, in some cases, the methylation proportion of the DNA marker fragments in the sample, that is, the number of methylated molecules/(the number of methylated molecules+the number of unmethylated molecules), can be calculated according to Ct values detected by fluorescence quantitative PCR of the sample, and then the methylation proportion, that is, the number of methylated molecules/(the number of methylated molecules+the number of unmethylated molecules) of the DNA marker fragments can be compared, and in some cases, statistical analysis and integration of each index are required to obtain a final judgment index.

In the context of the present application, the term "primer" refers to a macromolecule, which is stimulated to synthesize, upon initiation of nucleotide polymerization, a specific nucleotide sequence, which is hydrogen-bonded to a reactant, such a nucleic acid molecule being referred to as a primer. A "primer" is an artificially synthesized two oligonucleotide sequence, usually occurring and used in pairs, one primer being complementary to one DNA template strand at one end of the target region and the other primer being complementary to the other DNA template strand at the other end of the target region, which functions as a starting point for nucleotide polymerization, from the 3' end of which a nucleic acid polymerase can begin to synthesize a new nucleic acid strand. The exact length of the primer will depend on many factors, including temperature, primer source and use of the method. For example, in certain embodiments, the primer may range from 10 to 100 or more nucleotides.

In the context of the present application, the terms "patient", "subject" and the like are used interchangeably, and in embodiments of the present application "patient", "subject" is a human. In the context of the present application, the term "healthy person" refers to a population excluding ovarian cancer patients or patients at high risk of suffering from ovarian cancer, and does not merely represent a population without any definition of physical disease.

In the context of the present application, the terms "comprising," "including," and "containing" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

According to the embodiment of the application, through analyzing methylation levels of various genes in a TCGA database in ovarian Cancer tissues (Cancer) and paracancerous normal tissues (Normal adjacent tissues, NATs), the GPR120 gene on chromosome 10 of a human body and the DLGAP1 gene on chromosome 18 of the human body are found to show higher methylation levels (beta-value) in the Cancer tissues, and very low methylation levels are shown in the paracancerous normal tissues, so that target areas of the GPR120 gene and the DLGAP1 gene are found to be used as DNA methylation markers for diagnosing ovarian Cancer, and the DNA methylation markers can effectively distinguish ovarian Cancer patients from healthy people. In summary, embodiments of the present application provide a number of lines of evidence that indicate that the solutions provided by the current application are useful for the diagnosis of ovarian cancer.

Based on this, embodiments of the present application provide a biomarker for diagnosing ovarian cancer, comprising: a first nucleic acid fragment and/or a second nucleic acid fragment; the first nucleic acid fragment comprises the full length or partial region of the Chr10:93567151-93567551 positive strand of the GPR120 gene and the second nucleic acid fragment comprises the full length or partial region of the Chr18:3499055-3499455 negative strand of the DLGAP1 gene.

In certain embodiments, a biomarker for diagnosing ovarian cancer comprises a first nucleic acid fragment comprising the full length or partial region of the Chr10:93567151-93567551 positive strand of the GPR120 gene and a second nucleic acid fragment comprising the full length or partial region of the Chr18:3499055-3499455 negative strand of the DLGAP1 gene.

In certain preferred embodiments, a biomarker for diagnosing ovarian cancer comprises a first nucleic acid fragment and a second nucleic acid fragment; the nucleotide sequence of the first nucleic acid fragment is shown as SEQ ID NO.1, and the nucleotide sequence of the second nucleic acid fragment is shown as SEQ ID NO. 2. In these preferred embodiments, wherein the complete methylation sequence of the first nucleic acid fragment is shown in SEQ ID NO.3 and the complete unmethylation sequence of the first nucleic acid fragment is shown in SEQ ID NO. 4; the complete methylation sequence of the second nucleic acid fragment is shown as SEQ ID NO.5, and the complete unmethylation sequence of the second nucleic acid fragment is shown as SEQ ID NO. 6.

Based on the above, the embodiment of the application also provides a primer pair which can be used for detecting the methylation level of the biomarker fragment, thereby achieving the purpose of diagnosing ovarian cancer. The primer pair comprises a first primer pair and/or a second primer pair, wherein the first primer pair comprises a first methylation primer pair, the first methylation primer pair comprises a first upstream methylation primer and a first downstream methylation primer, and the nucleotide sequences of the first upstream methylation primer and the first downstream methylation primer are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8; the second primer pair comprises a second methylation primer pair comprising a second upstream methylation primer and a second downstream methylation primer, wherein the nucleotide sequences of the second upstream methylation primer and the second downstream methylation primer are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively.

In certain embodiments, primer pairs directed against the fully unmethylated sequence of the biomarker fragment are also designed for aiding detection of ovarian cancer, i.e., when the primer pair includes the first primer pair, the first primer pair further includes a first unmethylated primer pair including a first upstream unmethylated primer and a first downstream unmethylated primer, the nucleotide sequences of the first upstream unmethylated primer and the first downstream unmethylated primer are set forth in SEQ ID No.9 and SEQ ID No.10, respectively; when the primer pair comprises the second primer pair, the second primer pair further comprises a second unmethylated primer pair, the second unmethylated primer pair comprises a second upstream unmethylated primer and a second downstream unmethylated primer, and the nucleotide sequences of the second upstream unmethylated primer and the second downstream unmethylated primer are respectively shown as SEQ ID NO.13 and SEQ ID NO. 14.

In certain preferred embodiments, the primer pair for detecting the methylation level of the aforementioned biomarker fragment comprises a first primer pair and a second primer pair, and comprises all the primers shown as nucleotide sequences SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14.

Based on this, the embodiment of the application also provides a probe set for detecting the biomarker, which comprises at least one probe with nucleotide sequences shown as SEQ ID NO.17, SEQ ID NO.18 and SEQ ID NO. 19.

In certain preferred embodiments, the set of probes comprises three probes: a first probe with a nucleotide sequence shown as SEQ ID NO.17, a second probe with a nucleotide sequence shown as SEQ ID NO.18, and a third probe with a nucleotide sequence shown as SEQ ID NO.19, wherein the first probe and the second probe are used for detecting a first nucleic acid fragment of the biomarker, and the third probe is used for detecting a second nucleic acid fragment of the biomarker.

Based on this, the application provides a nucleic acid product for detection of ovarian cancer comprising the aforementioned primer pair and/or the aforementioned probe set.

In certain embodiments, the nucleic acid product further comprises a primer pair and a detection probe for detecting a reference gene.

Based on this, the application provides a kit for detection of ovarian cancer comprising the aforementioned nucleic acid product.

Based on this, the application provides the use of the aforementioned biomarker or the aforementioned primer pair or the aforementioned probe set or the aforementioned nucleic acid product or the aforementioned kit for the preparation of an ovarian cancer diagnostic product.

The technical solution and the technical effects achieved by the present application will be described in detail below by way of more specific examples.

Example 1

The present example screens and validates biomarkers for detection of ovarian cancer. This example shows that the GPR120 gene located on chromosome 10 and the DLGAP1 gene located on chromosome 18 show high methylation levels (β -value) in Cancer tissues, but very low methylation levels in Cancer tissues by analyzing the methylation levels of the individual genes in the TCGA database in ovarian Cancer tissues (Cancer) and in Cancer-associated normal tissues (Normal adjacent tissues, NATs). With hg38 as a reference genome, the physical position of the target region of the GPR120 gene (hereinafter referred to as "target region 1") is the positive strand Chr10:93567151-93567551, and the physical position of the target region of the DLGAP1 gene (hereinafter referred to as "target region 2") is the negative strand Chr18:3499055-3499455, and the methylation levels of the two target regions have significant differences in cancer tissues and paracancerous tissues, so that fragments of the GPR120 gene and DLGAP1 gene can be used as methylation molecular markers for diagnosing ovarian cancer.

In the embodiment, 68 ovarian cancer tissue samples and 68 paracancerous control tissue samples collected from a certain Wuhan hospital are used as training sets for pyrosequencing, and detection areas with sensitivity of 80% or more and specificity of 80% or more are selected: the nucleotide sequence of the target region 1 is shown as SEQ ID NO. 1; and target region 2, the nucleotide sequence is shown as SEQ ID NO.2, and the next verification is carried out, the specific process is as follows:

1. sample collection and processing

The tissue samples collected from hospitals were formalin-immersed, paraffin-embedded tissue samples, all samples were approved by the ethics committee during the collection process, and all volunteers signed informed consent.

Extraction of DNA

The collected tissue sample DNA was extracted using a blood/cell/tissue genomic DNA extraction kit (catalog number: DP 304) from Tiangen Biochemical technology (Beijing) limited, see kit instructions for specific procedures.

3. Conversion of bisulphite

The extracted DNA sample is subjected to bisulphite conversion, and the used nucleic acid conversion kit is a nucleic acid conversion reagent (Ehan mechanical preparation 20200843) of Wuhan Ai Misen life technology Co., ltd, and specific experimental operation is shown in the specification of the kit.

After the nucleotide sequences of the target region 1 and the target region 2 are subjected to the bisulfite conversion treatment, "C" in the DNA double strand is converted into "U", which is converted into "T" by the subsequent PCR reaction, but the bisulfite cannot cause the above-mentioned conversion of "C" of the DNA which has undergone methylation. The complete methylated and unmethylated sequences obtained after bisulfite treatment of target region 1 and target region 2 are shown in Table 1.

TABLE 1

PCR amplification

Designing two primer pairs for target region 1 and target region 2, respectively, one primer pair being for a bisulfite-treated methylated sequence; the other primer pair is directed to bisulfite treated unmethylated sequences, where methylated primers specifically amplify only methylated sequences and unmethylated primers specifically amplify only unmethylated sequences. According to the primer design principle, corresponding methylation primer pairs and unmethylation primer pairs are designed for different target areas, and specific information is shown in Table 2.

TABLE 2

The target region sequences amplified by the methylation primer pairs SEQ ID NO.7, SEQ ID NO.8 and the unmethylation primer pairs SEQ ID NO.9 and SEQ ID NO.10 comprise 13 CpG sites in SEQ ID NO.1, and the target region sequences amplified by the methylation primer pairs SEQ ID NO.11, SEQ ID NO.12 and the unmethylation primer pairs SEQ ID NO.13 and SEQ ID NO.14 comprise 17 CpG sites in SEQ ID NO. 2.

Adding SYBR Green PCR Mix methylation primer pairs and unmethylation primer pairs of target region 1 of GPR120 gene and target region 2 of DLGAP gene into DNA converted by bisulphite as templates, performing PCR amplification, and simultaneously adding detection primer pairs of internal reference gene ACTB, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.15, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.16, and the PCR reaction system is shown in the following table 3:

TABLE 3 Table 3

Component (A)	Dosage (mu L)
		SYBR Green PCR Mix	17.5
Methylation (unmethylation) upstream primer (10. Mu.M)	0.5
		Methylation (unmethylation) downstream primer (10. Mu.M)	0.5
ACTB upstream primer (10 mu M)	0.5
		ACTB downstream primer (10. Mu.M)	0.5
Template DNA	4
		Ultrapure water	Supplement to 25

The PCR reaction procedure is shown in Table 4 below:

TABLE 4 Table 4

5. Pyrosequencing

The PCR products were sent to sequencing company for pyrosequencing, and the sequencing peak pattern was analyzed based on methylation status of key CpG sites of target region 1 and target region 2 of pyrosequencing. Specifically, methylation of cytosine in a CpG site is classified into two types: i.e., methylated and unmethylated, where methylation is split into fully methylated and partially methylated, if the sequencing result of a cytosine at a CpG site shows that there is both a C and a T at the position of the cytosine, the site is considered partially methylated, and if more than 95% of the C's in CpG dinucleotide sites in an amplicon are methylated, the sample is considered methylated in this region.

6. Analysis of results

After the PCR reaction is completed, comparing the result of the amplified product sequencing with the pathological result, wherein the specific result of the comparison is shown in table 5 below, wherein the samples numbered 001-0068 are ovarian cancer positive tissue samples including high-grade serous ovarian cancer tissue, clear cell ovarian cancer tissue and serous ovarian cancer tissue samples, the samples numbered 0069-0138 are ovarian cancer negative tissue samples including high-grade serous ovarian cancer bypass tissue, clear cell ovarian cancer tissue and serous ovarian cancer tissue samples, and the methylation result of the target region 2 of the sample 0028 fall off, and statistics are not counted.

TABLE 5

The sensitivity and specificity of this target region was calculated by the methylation results for CpG sites in table 5 above, where sensitivity = methylation positive samples/samples with a positive pathological result; specificity = methylation negative samples/samples with negative pathology results, the results are shown in table 6 below:

TABLE 6

From the statistical results in table 6, it is known that the methylation level of the GPR120 gene target region 1 and DLGAP gene target region 2 in ovarian cancer tissues is significantly higher than that of ovarian cancer other normal tissues, and the methylation level of the target region 1 and the methylation level of the target region 2 are detected by using a methylation primer pair and a non-methylation primer pair, so that the sensitivity of diagnosing ovarian cancer is more than 83%, the detection specificity is more than 80%, and the requirements that the sensitivity of the target region 1 and the target region 2 is more than or equal to 80% and the specificity is more than or equal to 80% are met, so that the target region 1 and the target region 2 fragment can be used as biomarkers for further verification.

Example 2

In this example, 46 blood samples of ovarian cancer subjects and 46 blood samples of healthy subjects collected from a certain hospital in the armed state, all samples were approved by the ethical committee, all volunteers had signed informed consent prior to sample collection, and the performance of the above-screened target region 1 and target region 2 for diagnosis of ovarian cancer was further verified by pyrosequencing these blood samples, as follows:

Extraction of DNA

Centrifuging the collected blood sample, and separating blood cells and a plasma layer for later use; blood cells the genomic DNA of cells of each sample was extracted using a blood/cell/tissue genomic DNA extraction kit (catalog number: DP 304) from Tiangen Biochemical technology (Beijing) limited, see kit instructions for specific procedures; the plasma layer was subjected to plasma cfDNA extraction using a magnetic bead serum/plasma free DNA extraction kit (catalogue number: DP 709) from the company digen biochemical technology (beijing), the volume of plasma used being 1.5mL, see kit instructions for specific procedures.

2. Bisulphite conversion and purification treatments

The genomic DNA of each sample extracted was subjected to bisulfite conversion, and the nucleic acid conversion kit used was a nucleic acid purification reagent (Ehan Instrument preparation 20200843) from Kagaku Ai Misen Life technologies Co., ltd., see the kit instructions for specific experimental procedures, and after the conversion was completed, 30. Mu.L of purified water was used for elution.

PCR amplification and Sanger sequencing

The PCR amplification and Sanger sequencing methods were the same as in example 1.

4. Analysis of results

Analysis of the results was carried out in the same manner as in example 1, and the final detection results are shown in Table 7 below:

TABLE 7

From the results in table 7, it was found that the target region in which hypermethylation occurred in ovarian cancer was required to exclude interference in blood leukocytes, and the specificity of detection of healthy human blood leukocyte samples by target region 1 and target region 2 was 95.7% and 97.8%, respectively, indicating that target region 1 and target region 2 had good specificity in blood leukocyte samples. The target region of hypermethylation in ovarian cancer can be achieved by detecting a plasma sample, the detection specificity of the target region 1 and the target region 2 in a healthy human plasma sample is 95.7% and 97.8%, and the detection sensitivity of the target region 1 and the target region 2 in an ovarian cancer patient plasma sample is 80.4% and 82.6%, respectively, so that the target region 1 and the target region 2 have good effects for detecting the blood sample of the ovarian cancer patient and the healthy human.

Example 3

The embodiment also provides a method for detecting the methylation state of the GPR120 gene target region 1 and the DLGAP gene target region 2 in the plasma sample of the subject based on methylation fluorescence quantitative PCR, wherein the methylation state of the sample can be judged according to the Ct value, and further whether the blood sample is an ovarian cancer sample or a healthy sample is judged. After blinding the collected plasma sample in the test process of the embodiment, the test operator performs the test, and the result interpreter compares the obtained detection result with the pathological result according to the interpretation standard so as to investigate the clinical effectiveness of the biomarker for diagnosing ovarian cancer.

In the embodiment, 166 healthy human plasma samples are collected from a certain Wuhan hospital, 52 plasma samples with pathological diagnosis of ovarian cancer are included, and all patients do not receive radiotherapy or chemotherapy before operation; all samples were approved by the ethics committee, all volunteers signed informed consent, and all samples were anonymized. The present implementations diagnose ovarian cancer patients by detecting the methylation level of an ovarian cancer marker target region in a plasma sample of the subject.

The specific process is as follows:

1. extraction, transformation and purification of plasma sample DNA

Extracting the collected plasma sample DNA by using a nucleic acid extraction kit (Ehan mechanical equipment 20210836) provided by Wuhan Ai Misen life technology Co., ltd, wherein the specific operation is as follows; after the completion of the extraction, elution was performed with 50. Mu.L of purified water. The extracted plasma sample DNA was subjected to bisulfite conversion using a nucleic acid purification reagent (Ehan instruments 20200843) supplied by Wuhan Ai Misen Life technologies Inc., see kit instructions for specific procedures, and after completion of the conversion, eluted with 30. Mu.L of purified water.

2. Fluorescent quantitative PCR (qPCR) reaction

The detection probes were designed using the bisulfite converted polynucleotide sequences as templates, and specific detection primer and probe combinations are shown in table 8 below:

TABLE 8

The TaqMan probe method is used for PCR amplification of the target region of the clinical sample. When detecting the methylation level of a single target region, the PCR reaction tube is added with a necessary reaction component, a template, a methylation primer pair of the single target region, a detection probe and a detection primer pair of an internal reference gene ACTB (the nucleotide sequences are shown as SEQ ID NO. 15-16) and a detection probe (the nucleotide sequence is shown as SEQ ID NO. 20). In addition, the test sample also needs to be provided with a negative control, wherein the template of a PCR tube in the negative control is TE buffer solution, other components are the same as those of the test tube, the template of a positive control hole is 10 ³ copies/. Mu.L of plasmid containing the converted ACTB sequence and 10 ³ copies/. Mu.L of plasmid mixture (equal volume mixture) containing the DNA sequence of the target region after complete methylation and bisulphite conversion, and other components are the same as those of the test tube. If the methylation level of a combination of target regions is to be detected, a methylation primer pair and a detection probe of another target region are added to the PCR reaction tube in addition to the above components.

The detection probes are TaqMan probes, and the 5' end of the detection probes is a fluorescent reporter group, such as FAM, ROX, VIC, CY; the 3' end is a fluorescence quenching group, such as TAMRA, BHQ, MGB and the like. In the embodiment, the fluorescent reporter groups at the 5' ends of the detection probes SEQ ID NO.17 and SEQ ID NO.18 are ROX, the fluorescent reporter group at the 5' end of the detection probe SEQ ID NO.19 is VIC, and the fluorescent quenching groups at the 3' end are BHQ or BHQ1; the fluorescence report group at the 5 '-end of the detection probe of the reference gene ACTB is FAM, and the fluorescence quenching group at the 3' -end is MGB. The qPCR reaction system is shown in table 9 (if only a single target region is detected, no methylation primer pair and detection probe for another target region are added, the remaining volumes are made up with purified water):

TABLE 9

Component (A)	Specification of specification	Volume (mu L)
			Platinum II PCR buffer	5×	5
dNTPs	2.5MM each	3
			Methylation upstream primer for each target region	10μM	0.5
Methylation downstream primers for each target region	10μM	0.5
			Detection probes for each target region	10μM	0.5
ACTB gene upstream primer	10μM	0.5
			ACTB gene downstream primer	10μM	0.5
ACTB gene detection probe	10μM	0.5
			DNA polymerase	/	0.5
DNA of sample to be tested	/	4
			Purified water	/	Supplement to 25

TAQMAN PCR the procedure is shown in Table 10:

table 10

After qPCR reaction is finished, the baseline, which is usually a fluorescent signal of 3-15 cycles, is manually adjusted and a proper threshold is set, and the threshold is placed in an exponential amplification period.

3. Analysis of results

Analysis of the results of qPCR reactions required: ① The negative control PCR tube was not amplified; ② Positive control PCR tube

The method has obvious index increasing period, and the Ct value of the target area of the positive control PCR tube is between 26 and 30; ③ The Ct value of the reference gene of the sample to be detected is less than or equal to 34. If the positive control, the negative control and the reference gene all meet the requirements, the detection result of the sample to be detected can be analyzed and the result can be interpreted, otherwise, the detection must be carried out again when the experiment is invalid.

The positive judgment value of the plasma sample is 45, and if the Ct value of the amplified target area 1 is less than or equal to 45, the target area 1 is considered to be methylation positive; if the Ct value of amplified target region 1 is >45, target region 1 is considered methylation negative; the determination of the region 2 is the same as above; if at least one region in the region 1 or the region 2 in a certain sample to be detected is methylation positive, the sample is an ovarian cancer positive sample, namely the subject is judged to be ovarian cancer positive; if the region 1 and the region 2 in a certain sample to be tested are methylation negative, the sample is an ovarian cancer negative sample, namely the subject is judged to be ovarian cancer negative. According to the above criteria, specific detection results are shown in the following table 11:

TABLE 11

As is clear from the results shown in Table 11, the methylation level of target region 1 of GPR120 gene and target region 2 of DLGAP1 gene in plasma samples was measured by qMSP method, and thus the ovarian cancer was diagnosed. The sensitivity of detecting ovarian cancer ovarian plasma samples by the target region 1 of the GPR120 gene is 82.7%, and the specificity is 93.4%; the sensitivity of detecting ovarian cancer ovarian plasma samples by the target region 2 of DLGAP1 genes is 84.6%, and the specificity is 94.6%; and the sensitivity of the combined detection of the ovarian cancer plasma sample by adopting the target area 1 and the target area 2is improved to 86.5%.

According to the method for assisting early diagnosis of ovarian cancer by methylation detection of plasma free DNA (cfDNA), only 10mL of blood of a subject is required to be collected, and whether the patient suffers from ovarian cancer is assisted by detecting methylation signals of cfDNA; the distinguishing effect of the technology is superior to that of the traditional CA125 detection, particularly on a healthy human sample, the combined detection specificity of the target area 1 and the target area 2 can reach 92.8%, the proportion of benign patients which accept unnecessary invasive detection can be reduced to a great extent, and the infection risk and psychological stress brought by invasive detection are reduced.

In conclusion, the target region 1 of the GPR120 gene, the target region 2 of the DLGAP gene, the primer pair, the probe and the kit product for detecting the target region show the effect of diagnosing ovarian cancer, have higher detection sensitivity on ovarian cancer, have excellent detection specificity on a healthy human ovarian sample, have high detection accuracy and are more beneficial to early diagnosis.

The foregoing description is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any combination of the technical features of the foregoing embodiments may be arbitrarily combined, so that all possible combinations of the technical features of the foregoing embodiments are not described for brevity, and any changes or substitutions easily suggested by those skilled in the art are included in the scope of the present application.

Claims

1. A biomarker for diagnosing ovarian cancer, comprising:

And/or

2. The biomarker of claim 1, wherein the nucleotide sequence of the first nucleic acid fragment is set forth in SEQ ID No. 1; the nucleotide sequence of the second nucleic acid fragment is shown as SEQ ID NO. 2.

3. A primer pair for detecting a biomarker according to any of claims 1 to 2, comprising:

And/or

4. The primer pair of claim 3, wherein the first primer pair further comprises a first unmethylated primer pair comprising a first upstream unmethylated primer and a first downstream unmethylated primer, the nucleotide sequences of the first upstream unmethylated primer and the first downstream unmethylated primer being set forth in SEQ ID No.9 and SEQ ID No.10, respectively; the second primer pair further comprises a second unmethylated primer pair, the second unmethylated primer pair comprises a second upstream unmethylated primer and a second downstream unmethylated primer, and the nucleotide sequences of the second upstream unmethylated primer and the second downstream unmethylated primer are respectively shown as SEQ ID NO.13 and SEQ ID NO. 14.

5. A probe set for detecting a biomarker according to any of claims 1 to 2, comprising at least one of the probes having nucleotide sequences shown in SEQ ID No.17, SEQ ID No.18, SEQ ID No. 19.

6. The probe set of claim 5, wherein the fluorescent reporter group of the probes in the probe set is at least one of FAM, ROX, VIC, CY and the quencher group is at least one of TAMRA, BHQ, MGB.

7. A nucleic acid product for the detection of ovarian cancer, characterized in that it comprises a biomarker according to any of claims 1 to 2 and/or a primer pair according to any of claims 3 to 4 and/or a probe set according to any of claims 5 to 6.

8. The nucleic acid product of claim 7, further comprising a primer pair for detecting the reference gene ACTB.

9. A kit for the detection of ovarian cancer, comprising the nucleic acid product of claim 7 or 8.

10. Use of a biomarker according to any of claims 1 to 2 or a pair of substances according to any of claims 3 to 4 or a probe set according to any of claims 5 to 6 or a nucleic acid product according to any of claims 7 to 8 or a kit according to claim 9 for the preparation of a diagnostic ovarian cancer product.