CN113337614A - Marker, primer probe and kit for early screening and diagnosis of endometrial cancer - Google Patents
Marker, primer probe and kit for early screening and diagnosis of endometrial cancer Download PDFInfo
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Abstract
The invention discloses a marker, a primer probe and a kit for early screening and diagnosis of endometrial cancer, wherein the marker is a partial methylation region in four genes of CDO1, CELF4, HAND2 and HS3ST2, and a detection primer and a probe are designed aiming at the methylation region and are of a clamp ring type structure. The kit comprises the primer and the probe. According to the invention, the multi-methylation regions are screened and combined, the most appropriate methylation position is determined for joint diagnosis, and the sensitivity and specificity of detection on early endometrial cancer can be obviously improved. The kit is particularly suitable for early screening and diagnosis of endometrial cancer by taking cervical exfoliated cells as a sample, can detect the endometrial cancer even if the concentration of a DNA template is low, has the advantages of no sampling wound, high detection speed, higher sensitivity and specificity and the like, and can ensure the accuracy of the result so as to help realize the purposes of early discovery, early diagnosis and early treatment of the endometrial cancer.
Description
Technical Field
The invention relates to the technical field of nucleic acid in-vitro diagnosis, in particular to a marker, a primer and a kit for applying a specific gene methylation marker to early screening and early detection of endometrial cancer, and specifically relates to a marker, a primer and a kit for early screening and diagnosis of endometrial cancer.
Background
Endometrial cancer is an epithelial malignancy occurring in the endometrium, with a high incidence of age of 50-60 years, most commonly found in postmenopausal women, and has a tendency to become younger in recent years. According to the latest global cancer data report, the number of new cases of endometrial cancer of women in the world reaches 42 ten thousand in 2020, and the incidence rate of malignant tumors of women in the world is the sixth. In 2020, the number of new cases of endometrial cancer of Chinese women is 8 ten thousand, and the incidence rate of malignant tumors of Chinese women is ninth. In the united states, endometrial cancer is the fourth most common cancer that follows breast, lung and colorectal cancer. About 70% of patients with endometrioid carcinoma have been found to be confined to the uterus. Most endometrial cancer is localized lesion, the survival rate is relatively high, but symptoms such as early irregular vaginal bleeding and vaginal drainage are often ignored, and the opportunity of early diagnosis is lost. Data show that the disease fatality rate of endometrial cancer is increased more than the disease rate in recent years in China. Increased mortality may be associated with increased late-stage cases, high-risk types of pathology (e.g., breast cancer), advanced age at diagnosis, etc. U.S. monitoring, epidemiological and outcome (SEER) data show a better survival rate for younger patients, early cases, patients with low-grade lesions.
Most patients with endometrial cancer are sporadic, but about 5% of patients with endometrial cancer are hereditary, and the patients are characterized in that the onset age of the patients is 10-20 years less than the average age of patients with sporadic endometrial cancer. Female ringer syndrome is a high risk group for endometrial cancer with an incidence of up to 60%, suggesting that patients with ringer syndrome closely monitor the endometrium.
According to the current guidelines for diagnosis and treatment of endometrial cancer, vaginal ultrasound, a serum tumor marker CA125, cytological detection based on an endometrial cell sampler, and finally confirmed histopathological diagnostic curettage and hysteroscopic biopsy are mainly adopted clinically for the current method for early detection of endometrial cancer. The literature reports that endometrial cancer has certain ultrasound characteristic images. As long as the diagnosis skill and image characteristics of the endometrial cancer by ultrasound are well mastered, the endometrial cancer is diagnosed by ultrasound, but the sensitivity of the endometrial cancer in early stage is not high enough by ultrasound diagnosis, the sensitivity of the endometrial cancer is diagnosed by ultrasound, the specificity is 81.1%, the false negative rate is 40.6%, and the false positive rate is 18.9%. The detection of serum CA125 has certain value in early diagnosis of endometrial cancer and prediction of prognosis. However, the positive rates of serum CA125 in patients with endometrial cancer and benign uterine lesions are 24% and 20%, respectively, so that the detection of serum CA125 is insensitive to early diagnosis of endometrial cancer and relatively poor in specificity. Endometrial cells are not easy to fall off outside the menstrual period, and cancer cells falling off in the uterine cavity are easy to dissolve and denature and are not easy to identify after dyeing, so that the positive rate of cytological examination of vaginal falling off is not high. The early detection of endometrial cancer is carried out by combining an endometrial cell collector with a liquid-based cytology slice making technology, the sensitivity for diagnosing the atypical hyperplasia of the endometrium is 55 percent, the specificity is 82.6 percent, the sensitivity for diagnosing the endometrial cancer is 36.4 percent, and the specificity is 99.7 percent. Therefore, the method can be used for screening endometrium lesion of symptom people and high risk people by applying the liquid-based cytology examination of the endometrium cell collector, but the diagnostic technology sensitivity in the cytology examination process is poor, and the sampling has certain traumatism. Diagnostic curettage and hysteroscopic biopsy are essential methods for the definitive diagnosis of endometrial cancer, and given that endometrial biopsy may have about 10% false negatives, if endometrial cancer is highly suspected or with typical symptoms, the negative endometrial biopsy should be sectioned again under anesthesia, and cervical canal scratched to reduce missed diagnosis. For patients with continuous or repeated vaginal bleeding without definite endometrial lesion, hysteroscopy is helpful for judging the benign and malignant degree of the endometrial lesion, but has great harm to human bodies, is not suitable for early screening of the endometrial lesion, and can only be used for final pathological confirmation. Thus, there is a need for an objective, reproducible, and highly specific assay to better discern the potential for endometrial cancer. At present, an effective endometrial cancer screening means is not available, and a noninvasive screening method can be explored. At present, the incidence rate of endometrial cancer in China shows an increasing trend every year, and a new screening means is still slow.
DNA methylation is one of epigenetic modifications of cytosine at the 5' end of DNA, and is often associated with gene silencing. Because DNA methylation remains relatively stable over long periods of time and is present in samples (e.g., paraffin sections), DNA methylation is the most useful apparent marker for human disease research. In early screening-related research and applications, methylation marker detection is currently generally accepted as the most feasible detection scheme, because DNA methylation markers have better stability than mRNA, protein or metabolite markers, and are convenient for stable detection; compared with DNA markers, the DNA marker has better diversity and can reflect the influence of the change of internal and external environments of a body. Methylation is a modification on DNA that finely regulates gene transcription, essentially showing hypermethylation that reduces gene expression and hypomethylation that increases gene expression. The methylation level and the pattern are changed to cause the expression of the cancer suppressor gene to be switched off or the expression of the proto-oncogene to be activated, so that the detection of the DNA methylation level of a specific site can be used for predicting the growth condition of an individual tumor, thereby realizing the aim of early screening and early diagnosis of the tumor.
In recent years, DNA methylation has attracted increasing attention for basic research and clinical transformation. A large number of channels are found to be regulated by epigenetics, and methylation big data based on a chip technology and a second-generation sequencing technology are generated, and a plurality of existing researches show that the DNA methylation level in body fluid is changed, so that the DNA methylation level can be used for early diagnosis, prognosis evaluation and curative effect evaluation of cancer; some leading edge work has begun to transform and enter clinical applications. DNA methylation has been clinically applied to early screening of intestinal cancer, and is represented by cologuard (large intestine guard) of exact sciences corporation in the United states and Nuohui and Corimen in China. Although the early-screening tumor diagnosis of DNA methylation of other types of cancers is also relevant research by enterprises at home and abroad, the early-screening tumor diagnosis is still in the research stage as a whole, and can be applied to clinic and has a definite diagnostic value, wherein the Foundation Medicine of the U.S. company carries out research and research on partial data in the methylation direction of multiple solid tumors (lung cancer, breast cancer, stomach cancer, esophageal cancer, colorectal cancer, liver cancer, bile duct gallbladder cancer, pancreatic cancer, gastrointestinal stromal tumor, head and neck cancer, cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, kidney cancer, melanoma and the like). In 2016, Huang et al screened 180 related genes by using cervical secretions of 50 cases of endometrial cancer, 40 cases of uterine fibroids and 56 cases of healthy people as research objects, found 14 genes hypermethylated in the endometrial cancer, wherein three genes, namely BHLHE22, CDO1 and CELF4, are particularly outstanding in performance, the sensitivity of the genes is between 83.7% and 96%, the specificity of the genes can also reach 78.7% to 96.0%, and meanwhile, the sensitivity of the genes can reach 91.8% while the specificity of the genes is kept at 95.5% by carrying out combined detection on every two genes. In 2014, Nicolas et al collected 148 cases of endometrial cancer specimens and 23 cases of benign endometrial specimens, studied 1500 probes covering 807 genes, screened out 8 potential genes (ADCYAP 1, ASCL2, HS3ST2, HTR1B, MME, NPY, and SOX 1), and could have the potential ability to distinguish the benign and malignant endometrium. Allison et al studied the methylation of 27,000 CpG islands in 64 endometrial tissue samples and 23 control samples in 2013 and found that the HAND2 gene is one of the most common hypermethylated genes in endometrial cancer, while the role of HAND2 was again demonstrated in a subsequent study with 272 women, and that methylation of this gene is one of the important factors in the development of endometrial lesions.
DNA methylation occurs primarily in the promoter region of genes and is often closely associated with inactivation of expression of cancer suppressor genes. The current methods applied to gene methylation detection mainly include: methylation-specific PCR (MSP), Bisulfite Sequencing PCR (BSP), and High Resolution Melting curve method (HRM). Methylation-specific PCR relies primarily on the binding of primers to a target template for PCR amplification to detect methylation sites; the bisulfite sequencing method relies on sequencing primers to perform PCR amplification, and on the basis, subsequent sequencing is performed to realize detection of methylation sites; the high resolution dissolution curve method distinguishes between methylated and unmethylated cases primarily by the change in dissolution temperature due to changes in the CG content of the sample. Each method has respective characteristics, the BSP method has higher result accuracy, is easy to visually judge and read, but has lower sensitivity, more complicated operation and high cost; the HRM method has relatively low sensitivity and slightly complex result analysis; the MSP method has high detection sensitivity, relatively low requirement on samples, short detection time, low cost and easy interpretation of results.
Non-invasive and low cost are desirable features for early screening of cancer. For detection of endometrial cancer, negative ultrasound or cytological detection based on an endometrial sampler, diagnostic uterine curettage and the like are mainly adopted clinically, on one hand, the detection sensitivity is low, and on the other hand, the direct collection of endometrial cells has certain invasiveness.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a marker, a primer, a probe and a kit for early screening and diagnosing endometrial cancer, which have the advantages of high detection accuracy, convenience and rapidness in operation and capability of providing a reliable reference basis for clinical diagnosis of endometrial cancer. The cervical exfoliated cells can be used as a detection sample, cannot damage a patient, and are easier to accept by the patient.
The detailed technical scheme of the invention is as follows:
in a first aspect, the present invention provides a set of markers for early screening and diagnosing endometrial cancer, which are selected from at least one methylation region of any of the following genes in each of target genes CDO1, CELF4, HAND2 and HS3ST 2:
CDO1 gene: chr5:115805377-115805492, Chr5:115806258-115806356, Chr5: 115806540-115806618;
CELF4 gene: chr18:37243195-37243249, Chr18:37244217-37244299, Chr18: 37244683-37244784;
HAND2 gene: chr4:173526387-173526445, Chr4:173527003-173527077, Chr4: 173527734-173527835;
HS3ST2 gene: chr16:22813832-22813912, Chr16: 22814061-22814120, and Chr16: 22814470-22814540.
The four marker genes (or called target genes) and the corresponding optimal methylation regions can be finally screened out by jointly screening the endometrial cancer related gene types and each gene methylation region, the interpretation threshold value can be determined by the complementarity of the methylation results of the marker genes, the results of the methylation regions are combined with each other and used for early detection of the endometrial cancer, the result accuracy is very high, and auxiliary diagnosis reference can be provided for clinicians.
Alternatively or preferably, in the marker for early screening and diagnosing endometrial cancer, the following methylation regions in each gene selected from the group consisting of target genes CDO1, CELF4, HAND2 and HS3ST 2:
CDO1 gene: chr5:115806540 and 115806618;
CELF4 gene: chr18: 37244217-37244299;
HAND2 gene: chr4: 173527734-173527835;
HS3ST2 gene: chr16: 22813832-22813912.
The combined detection effect is best.
In a second aspect, the present invention further provides a set of detection primers for early screening and diagnosis of endometrial cancer, which are used for correspondingly detecting the methylation state of the methylation region of the marker gene, wherein the primers are designed to adopt a clasp type structure, a sequence with a length of 5-10bp and complementary pairing with the 3 ' end is added to the 5 ' end of a primer nucleotide sequence, the sequence does not contain CG sites, the last two bases at the end of the 3 ' end are CG, and the Tm value of a double-stranded binding region of the primers should be consistent with the annealing temperature in a PCR reaction system.
In the design of the primer, an added sequence with the length of 5-10bp does not contain a CG site, so that the binding is more compact when a snap ring is formed. The primer sequence adopts a clamp ring type design structure, firstly, no primer dimer is generated, and during the annealing process, the primer dimer is preferentially kept to form a double-stranded structure and not form double strands with other primers, so the primer dimer is not formed. Secondly, the primer has high specificity, and because the primer has a clamp ring structure, the combination free energy of the primer and a methylated target sequence needs to be larger than the ring free energy formed by the primer, when mismatched bases exist at the 3' end of the primer, the primer containing the clamp ring structure is hardly combined with a non-methylated sequence, and the primer amplification has better specificity. And thirdly, the kit has high sensitivity, and because the used sample is cervical exfoliated cells, the quantity of the DNA template is small, the content of methylated DNA in the template is low, and the primer designed for the clasp type structure can only be singly matched with a methylated target sequence, so that the amplification efficiency of the primer is greatly improved. The design and synthesis process of the primer does not need to add other additional modifications, the synthesis is simple, and the synthesis can be completed only by a commonly used synthesizer.
The primers keep high specificity of primer amplification due to a clamp ring type design structure, so that the amplification efficiency among the primers cannot generate interference, and the primers have obvious advantages for the amplification of multiple primers. The invention adopts 4 target genes and one reference gene to amplify in a reaction system, and the mutual influence of the amplification efficiency among gene primers does not exist.
Optionally or preferably, the detection primer for early screening and diagnosing endometrial cancer has a nucleotide sequence shown as follows:
CDO1 gene:
the detection primers corresponding to Chr5:115805377-115805492 are SEQ ID NO 1-2,
the detection primers corresponding to Chr5:115806258 and 115806356 are SEQ ID NO. 4-5,
the detection primers corresponding to Chr5:115806540 and 115806618 are SEQ ID NO 7-8;
CELF4 gene:
the detection primers corresponding to Chr18:37243195 and 37243249 are SEQ ID NO 10-11,
the detection primers corresponding to Chr18:37244217-37244299 are SEQ ID NO 13-14,
the detection primers corresponding to Chr18:37244683 and 37244784 are SEQ ID NO 16-17;
among the HAND2 genes:
the corresponding detection primers of Chr4:173526387 and 173526445 are SEQ ID NO. 19-20,
the corresponding detection primers of Chr4:173527003-173527077 are SEQ ID NO. 22-23,
the corresponding detection primers of Chr4:173527734-173527835 are SEQ ID NO. 25-26;
HS3ST2 gene:
the corresponding detection primers of Chr16:22813832 and 22813912 are SEQ ID NO 28-29,
the corresponding detection primers of Chr16: 22814061 and 22814120 are SEQ ID NO. 31-32,
the corresponding detection primers of Chr16: 22814470 and 22814540 are SEQ ID NO 34-35.
In a third aspect, the invention also provides a group of detection probes for early screening and diagnosis of endometrial cancer, which are used for correspondingly detecting the marker genes, wherein the nucleotide sequence of the detection probes is designed to adopt a snap ring type structure, a section of sequence which is 3-6bp long and is complementarily paired with a 3-4bp region close to the tail end of the 3 'end is added at the 5' end of the nucleotide sequence, the section of sequence does not contain CG sites, the Tm value of a double-stranded binding region of the probes is higher than the annealing temperature in a PCR reaction system by 2-15 ℃, and the binding free energy of the nucleotide sequence of the probes and the marker gene sequence is higher than the snap ring structure formed by the probes; the 5 'end of the detection probe is marked with a fluorescent group, the 3' end of the detection probe is marked with a quenching group, and different marker gene detection probes in the same detection system are marked with different fluorescent groups.
Fluorophores include, but are not limited to: FAM, ROX, CY5, HEX, quenching groups including but not limited to BHQ1, BHQ 2.
The target gene detection probes marked by different fluorescent channels can be placed in a tube for reaction, so that the optimal amplification efficiency of different target genes in a sample is ensured, the fluorescent curve is a standard S-shaped amplification curve, and the fluorescent curve keeps the consistent trend compared with the single amplification of each gene.
The design of the probe also adopts a clamp ring type design structure, so that the fluorescent group and the quenching group can be close to each other, and no fluorescence is generated when the probe forms a ring structure. Meanwhile, the probe sequence contains 2-5 CG sites for specifically recognizing methylated sequences. A3-6 bp sequence is added at the 5' end of the probe to form base sequence complementation with a 3-4bp region close to the 3 end tail end, the complementation region does not contain CG sites, the Tm value of a double-chain combination region of the probe is 2-5 ℃ higher than the annealing temperature in a reaction system, the Tm value is required to be increased by 5-10 ℃ along with the increase of the CG sites, and the specific requirement is determined according to the screening result of the probe. In the process of designing the probe, the free energy of binding between the probe sequence and the target sequence is higher than that of a snap ring structure formed by the probe sequence itself.
The advantages of this probe design are: firstly, due to the formation of a clasp type structure, the distance between a fluorescent group and a quenching group of a sequence is relatively short, the fluorescence quenching effect is better, and extra fluorescence cannot be generated, so that the fluorescence background is low. And secondly, the probe has high specificity, on one hand, the probe is of a clamp ring type design structure, the sequence is difficult to combine with a non-methylated sequence, and on the other hand, the probe contains 2-5 CG sites, so that the probe sequence is ensured to be only combined with a matched methylated sequence. Thirdly, the high sensitivity, the fluorescence can be detected only by opening the probe ring structure and combining the probe ring structure with the methylated sequence and separating the fluorescent group from the quenching group. And fourthly, the probe sequences are suitable for multiple amplification reactions, snap ring structures are respectively formed among the probe sequences, complementary pairing combination is difficult to carry out among the probe sequences, primer dimers cannot be generated, and combination of the probe sequences and target sequences cannot be influenced.
Alternatively or preferably, the nucleotide sequence of the detection probe for early screening and diagnosing endometrial cancer is as follows:
CDO1 gene detection probe: 9, SEQ ID NO;
CELF4 gene detection probe: 15, SEQ ID NO;
HAND2 gene detection probe: 27, SEQ ID NO;
HS3ST2 gene detection probe: SEQ ID NO 30.
In a fourth aspect, the present invention provides a detection kit for early screening and diagnosing endometrial cancer, which comprises any one of the detection primers described above, and any one of the detection probes described above.
Optionally or preferably, the detection kit for early screening and diagnosing endometrial cancer further comprises an internal reference gene detection primer and a probe, wherein the internal reference gene is GAPDH, the nucleotide sequence of the internal reference gene detection primer is shown in SEQ ID NOs 37-38, the nucleotide sequence of the internal reference gene detection probe is shown in SEQ ID No. 39, the 5 'end of the nucleotide sequence of the internal reference gene detection probe is labeled with a fluorescent group, the 3' end of the nucleotide sequence of the internal reference gene detection probe is labeled with a quenching group, and the fluorescent group labeled by the internal reference gene detection probe in the same detection system is different from the fluorescent group labeled by the marker gene detection probe.
Optionally or preferably, the detection kit for early screening and diagnosing endometrial cancer further comprises a PCR reaction solution and a sample methylation pretreatment reagent; the sample is a cervical exfoliated cell, and the sample methylation pretreatment reagent comprises a cell genome DNA extraction reagent and a DNA bisulfite conversion reagent.
The sample pretreatment plays an important role in the whole kit, and is directly related to the subsequent PCR amplification effect.
The kit is particularly suitable for taking cervical exfoliated cells as samples. The currently clinically common sampling method is based on collecting endometrial samples with an endometrial cell sampler or obtaining endometrial tissue in the form of a uterine curettage, which is invasive to the patient and may give the patient some invasiveness. The cervical exfoliated cell sample adopted in the kit detects cells exfoliated from the endometrium area to the cervical part, so that the content of endometrial cells is low, but the kit is simpler and more noninvasive and easy to obtain for a sample source. Because the content of endometrial cells in cervical exfoliated cells is low, the kit is particularly important for the sample pretreatment reagent and the subsequent PCR reaction solution contained in the kit, and whether the change of DNA methylation in the endometrial cells can be detected or not is determined.
When the sample methylation pretreatment reagent is used, two processes are involved, one is a process for extracting genomic DNA, and the other is a process for converting bisulfite, wherein one process has problems, and the final result of the process is changed greatly.
The cell genome DNA extraction reagent takes the cervical exfoliated cells as samples, preferably adopts a genome DNA extraction kit (nucleic acid extraction or purification reagent (Beijing original poly-rice biotechnology, Inc., Beijing Dajizhi 20210020)) developed by the applicant to extract DNA from the cervical exfoliated cells, and the extraction/purification kit has great improvement on the yield of the DNA and the purity of the DNA by contrast screening in the development process, extracts 2mL of the cervical exfoliated cell preservative fluid, the total amount of the DNA is between 4 and 8 mu g, and the OD260/280 is between 1.9 and 2.0, and keeps better yield and purity.
After the DNA extraction is completed, DNA bisulfite conversion experiments are performed, and DNA bisulfite conversion reagents are needed. It is also preferable to use a bisulfite conversion kit (methylation detection sample pretreatment kit (Beijing-originated poly rice biotechnology limited, Kyoto Med. 20200110)), which is autonomously developed by the applicant, and the conversion process compares the main indexes: firstly, the conversion rate of bisulfite can convert C in some sequences into U, and secondly, the purification efficiency after conversion is the final yield of bisDNA. When the DNA bisulfite conversion reagent is used, the conversion efficiency of bisulfite is 99.8 percent, the purification efficiency is 99 percent, and high-quality bisDNA is provided for the subsequent PCR amplification reaction.
Optionally or preferably, in the detection kit for early screening and diagnosing endometrial cancer, each PCR reaction solution comprises 0.5 to 1 μ L of Taq DNA polymerase with methylation characteristic at a concentration of 1U/μ L, 2 to 5 μ L of dNTPs with a concentration of 10 mM, and2 to 5 mM of Mg2+2-6 μ L, 10 XDNA polymerase buffer 5 μ L and purified water to make up 15 μ L.
In the PCR reaction system, the important component is Taq polymerase with methylation specificity, and the PCR reaction system mainly has the following advantages: the template sequence after the bisulfite conversion is amplified, the converted sequence can be specifically identified, and the amplification efficiency of the primer on the converted sequence is improved. When the amount of the enzyme is insufficient, the amplification efficiency is reduced, and when the amount of the enzyme is too large, nonspecific amplification is easily caused, so that the subsequent PCR amplification result can be significantly influenced by the amount of the enzyme. In addition, dNTPs and Mg in the system2+The ratio of 10 XDNA polymerase buffer is also directly related to the amplification efficiency of the combination of primer and probe.
The PCR reaction system is specially aimed at bis-DNA amplification after bisulfite conversion, and comprises multiple primer probes, so that the selection of PCR reaction liquid is particularly important, the amplification efficiency of each gene primer probe in the system is similar to that of corresponding single amplification, the primers or probes in the system are ensured not to be mutually interfered, and the amplification effect of each group of primer probes is fully exerted. The Taq polymerases with different methylation specificities and the proportions of the Taq polymerases with other components need to be screened and verified, so that the optimal amplification efficiency of the whole multiplex amplification system is ensured.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention screens four marker genes (target gene and target gene) of CDO1, CELF4, HAND2 and HS3ST2, determines the optimal methylation region in each gene, and can be combined with each other for early detection of endometrial cancer. The screened areas not only comprise promoter areas of genes, but also comprise coding areas of the genes, and due to the variety diversity of endometrial cancer, the multiple gene methylation areas are selected for combined detection to form functional complementation, so that the sensitivity of endometrial cancer detection is obviously improved, and the screened areas have high specificity on normal and endometrium benign tumors. The detection composition detects possible patients with gynecological malignant tumors in advance by a molecular epigenetic means and a methylation detection technology, has high result accuracy, and can provide auxiliary diagnosis reference for clinicians to prevent and treat the gynecological malignant tumors in advance.
2. The detection primers and the probes are designed aiming at the specific methylation sites of the four combined marker genes, and the clamp ring type design structure is adopted, so that the methylation template can be identified doubly, the detection sensitivity and specificity are obviously improved, the detection accuracy is improved, and the detection error is reduced. The primer and the probe can obtain accurate detection results from a small amount of samples, and are more suitable for clinical application.
The detection system formed by all primers and probes can also adopt a multi-gene multi-channel fluorescence detection means, five fluorescence probes are used for marking, the detection system accurately identifies a methylation sequence through a specific primer probe and an optimized special methylated DNA Taq polymerase, and accurately detects methylation sites of CDO1, CELF4, HAND2 and HS3ST2 genes, the detection of the multi-gene methylation sites is completed in batches, the detection method is simple to operate and intuitive to interpret, results are obtained within 8 hours, a universal fluorescence quantitative PCR instrument can meet the detection requirements, the whole set of experiment process adopts a one-station type fully-closed form, the operation is simpler and more convenient, and the possibility of cross contamination is avoided.
3. A sample methylation pretreatment reagent and a PCR reaction solution in the kit respectively provide guarantee for DNA extraction and pretreatment of cervical exfoliated cells and subsequent PCR reaction. At present, the phenomenon of non-specific amplification (false positive result) occurs in PCR amplification, the most mainstream bisulfite conversion technology at present is used for converting the extracted sample, the method is limited by the current bisulfite conversion technology, about 80% of target genome is likely to be lost, and a certain probability of unconverted template is also likely to cause false positive result. The invention adopts a clamp ring structure in the design of a target gene primer probe, increases the sensitivity and specificity of amplification, and optimizes the sample methylation pretreatment (including DNA extraction and bisulfite conversion) to improve the conversion yield of the final bisDNA.
4. The kit is combined with a specific primer probe, a sample pretreatment reagent, DNA polymerase of PCR reaction liquid and the like, so that the kit keeps high sensitivity when in use, has good primer probe specificity and high amplification efficiency, has very good detection rate for a template with low concentration, and is very sensitive to the detection of early endometrial cancer. The high sensitivity of the detection of the kit is suitable for early detection of endometrial cancer.
Drawings
FIG. 1 is a ROC curve for screening methylation regions for each of the genes CDO1, CELF4, HAND2, and HS3ST 2;
FIG. 2 is a ROC curve for the combined detection of CDO1, CELF4, HAND2, and HS3ST 2.
Detailed Description
The technical solutions of the present invention are explained and illustrated in detail below with reference to the accompanying drawings and preferred embodiments so that those skilled in the art can better understand the present invention and implement the present invention.
Example 1
Detection tests of the kit for detecting endometrial cancer related genes CDO1, CELF4, HAND2 and HS3ST2 methylation.
The specific nucleotide sequences of the primers and probes used are shown in the following table:
CDO1-F1 | ctaaatTTTTTTTTTTTTTTTATTTAGCG | SEQ ID NO:1 |
CDO1-R1 | aatttcaccAGAGAGGGGGTGAAATTGC | SEQ ID NO:2 |
CDO1-FP1 | FAM-tataatTAGCGTCGCGAATTATA-BHQ1 | SEQ ID NO:3 |
CDO1-F2 | aaccaaGTTTAAAGTGATTGGTTCG | SEQ ID NO:4 |
CDO1-R2 | cttccacctGGTTATTTTAGGTGGAAGGC | SEQ ID NO:5 |
CDO1-FP2 | FAM-acccTTAGTCGCGGGGTTGGT-BHQ1 | SEQ ID NO:6 |
CDO1-F3 | ataacGTTTATATTTTTAAGTTATCG | SEQ ID NO:7 |
CDO1-R3 | ctaatcatcTGGGAGATGATTAGGC | SEQ ID NO:8 |
CDO1-FP3 | FAM-catctATTTCGGGCGCGGAGATGCGG-BHQ1 | SEQ ID NO:9 |
CELF4-F1 | aaaaaaaATGTAGTTTTTTTTTTTTCG | SEQ ID NO:10 |
CELF4-R1 | aaaaaaatcTAAATTTGATTTTTTTGC | SEQ ID NO:11 |
CELF4-FP1 | ROX-aatccAATGCGCGTTCGGATTTTCG-BHQ2 | SEQ ID NO:12 |
CELF4-F2 | attccatGTATATAAAGATGGTTACG | SEQ ID NO:13 |
CELF4-R2 | ctaattcaaTTGATATTGAATTAGGC | SEQ ID NO:14 |
CELF4-FP2 | ROX-atctaTAACGGGTTCGGTAGTAGTT-BHQ2 | SEQ ID NO:15 |
CELF4-F3 | cccaaaaTAAGATTGGGTTTTGGGCG | SEQ ID NO:16 |
CELF4-R3 | taaactttcGTAGGGGTAGAAAGTTTAGC | SEQ ID NO:17 |
CELF4-FP3 | ROX-cttcTAGCGGGCGTCGATGAAGAGA-BHQ2 | SEQ ID NO:18 |
HAND2-F1 | attataaaAAATAATAGTATTTATAATCG | SEQ ID NO:19 |
HAND2-R1 | actcaataaGATAAATTTTATTGAGTGC | SEQ ID NO:20 |
HAND2-FP1 | CY5-tcctcTGTTCGTTCGGAGGATTTA-BHQ2 | SEQ ID NO:21 |
HAND2-F2 | acaataaaaTTAAAAAGTTTTATTGTCG | SEQ ID NO:22 |
HAND2-R2 | caaaatttcGTTGATGTAGAAATTTTGGC | SEQ ID NO:23 |
HAND2-FP2 | CY5-aaaaGTTCGAGGCGTCGTTTTTGT-BHQ2 | SEQ ID NO:24 |
HAND2-F3 | ctattaatGGATTTAGAGTATTAATAGCG | SEQ ID NO:25 |
HAND2-R3 | aaaccaaTATATTGATTATTGGTTTGC | SEQ ID NO:26 |
HAND2-FP3 | CY5-caatTTCGTCGAATTGCGCG-BHQ2 | SEQ ID NO:27 |
HS3ST2-F1 | caaacacccATTAGGGTAGGGTGTTTGCG | SEQ ID NO:28 |
HS3ST2-R1 | accaccccGATTTGTGGGGGTGGTGC | SEQ ID NO:29 |
HS3ST2-FP1 | HEX-aaatTCGGCGCGATTTCGATTTGGA-BHQ1 | SEQ ID NO:30 |
HS3ST2-F2 | cccccactGTAAGAGAGTGGGGGCG | SEQ ID NO:31 |
HS3ST2-R2 | aatccaatTAAATTTTTTATTGGATTGC | SEQ ID NO:32 |
HS3ST2-FP2 | HEX-ccccCGGCGCGGGTTCGGGGGATT-BHQ1 | SEQ ID NO:33 |
HS3ST2-F3 | aaaactacTTTTGGTTAGTAGTTTTCG | SEQ ID NO:34 |
HS3ST2-R3 | atcccaatTTTGATGATATTGGGATGC | SEQ ID NO:35 |
HS3ST2-FP3 | HEX-aaaaAGCGTTACGCGAGTTTTTTAG-BHQ1 | SEQ ID NO:36 |
GAPDH-F | AGGTTAAATATAGTTGTTGA | SEQ ID NO:37 |
GAPDH-R | CAACCCAAACCCCCAAC | SEQ ID NO:38 |
GAPDH-FP | Joe-TAGTTGGGGGTTTGGGTT-BHQ1 | SEQ ID NO:39 |
note: f represents a forward detection primer, R represents a reverse detection primer, and FP represents a detection probe. In this table, the probe sequences shown have been labeled with a fluorophore and a quencher.
The components of the kit without the sample pretreatment reagent are as follows:
60 examples of endometrial cancer samples with known and definite pathological information results were selected: 15 cases were identified as endometrioid carcinoma, 15 cases of endometrial mucinous carcinoma, 15 cases of endometrial serous carcinoma, 15 cases of endometrial clear cell carcinoma; 40 cases are benign samples of the endometrium. The above samples are obtained by reserving the cervical exfoliated cells.
First, sample methylation pretreatment
The sample methylation pretreatment reagent comprises a cell genome DNA extraction reagent and a DNA bisulfite conversion reagent.
1. Genome DNA extraction and DNA quality monitoring were carried out on 100 samples of endometrium good/malignant cervical exfoliated cell samples by using a genome DNA extraction kit (nucleic acid extraction or purification reagent (Beijing-originated Poa Biotech Co., Ltd., Beijing Dajiu No. 20210020)) developed by the company as a cell genome DNA extraction reagent. The total amount of DNA is between 4 mu g and 8 mu g, OD260/280 is between 1.9 and 2.0, and better yield and purity are kept.
2. A bisulfite conversion kit (methylation detection sample pretreatment kit (Beijing-originated Poa Biotechnology Co., Ltd., Beijing Dajiu No. 20200110)) independently developed by the company is adopted as a DNA bisulfite conversion reagent, so that the extracted DNA is subjected to bisulfite conversion, unmethylated cytosine (C) in the DNA is converted into uracil (U), and methylated cytosine (C) is not changed, and the converted bis-DNA is obtained. In the embodiment, the conversion efficiency of the bisulfite is 99.8 percent, which is higher than that of most bisulfite conversion kits on the market.
Secondly, performing fluorescent quantitative PCR amplification on bis-DNA
3. Preparing PCR reaction liquid and primer probe mixed liquid;
PCR reaction solution (15. mu.L/person)
Components | One part addition amount (mu L) |
DNA polymerase with methylation characteristics (1U/. mu.L) | 0.85 |
dNTPs(10mM) | 4 |
Mg2+(2-5mM) | 3 |
10 XDNA polymerase buffer | 1.5 |
Purified water | Make up to 15. mu.L |
Primer probe mixed solution (5 mu L/person)
Components | One part addition amount (mu L) |
CDO1/CELF1/HAND2/HS3ST2-F(100μM) | 0.2-0.8 |
CDO1/CELF1/HAND2/HS3ST2-R(100μM) | 0.2-0.8 |
CDO1/CELF1/HAND2/HS3ST2-FP(100μM) | 0.2-0.4 |
GAPDH gene-F (100. mu.M) | 0.05 |
GAPDH gene-R (100. mu.M) | 0.05 |
GAPDH gene-FP (100. mu.M) | 0.05 |
Purified water | Make up to 5. mu.L |
4. Sample application
5 mul of negative and positive quality control substances and the transformed Bis-DNA clinical sample are respectively added into the prepared system. Carrying out PCR reaction under the following conditions: pre-denaturation at 96 ℃ for 5 min; denaturation at 94 ℃ for 15s, annealing and extension at 60 ℃ for 35s, and 45 cycles; keeping at 25 deg.C for 10 min.
5. The amplification procedure was as follows:
step 1: pre-denaturation at 96 ℃ for 5 min;
step 2: denaturation at 94 ℃ for 15s, annealing and extension at 60 ℃ for 35s, and 45 cycles;
step3:25℃,10min;
signal Collection, FAM, HEX, ROX, Joe and CY5 signals were collected at 60 ℃.
6. Interpretation of results
(1) The internal standard channel has an S-shaped amplification curve, and the Ct value is less than or equal to 32.2, so that the result is effective;
(2) the Δ Ct values for the 4 genes are:
ΔCt(CDO1)=Ct(CDO1)-Ct(GAPDH);
ΔCt(CELF4)=Ct(CELF4)-Ct(GAPDH);
ΔCt(HAND2)=Ct(HAND2)-Ct(GAPDH);
ΔCt(HS3ST2)=Ct(HS3ST2)-Ct(GAPDH)。
(3) and (3) integrating the delta Ct values of the 4 genes, determining the boundary values and the performances (including specificity, sensitivity, negative predicted values and positive predicted values) of a plurality of methylation regions of the target gene according to the ROC curve, determining the optimal methylation region and the interpretation modes of the 4 target genes.
7. Analysis of detection results
The reaction system of the kit is used for detecting 100 samples in total, wherein the samples comprise 60 endometrial cancer samples and 40 endometrial benign samples.
Comparing the clinical and pathological results, in 100 samples of cervical exfoliated cells,
the positive rate of CDO1 (Chr 5: 115805377-115805492) in endometrial cancer was 58.3% (35/60), the specificity in benign samples was 82.5% (33/40), and the ROC area was 0.728;
the positive rate of CDO1 (Chr 5: 115806258-115806356) in endometrial cancer was 50% (30/60), the specificity in benign samples was 92.5% (37/40), and the ROC area was 0.714;
the positive rate of CDO1 (Chr 5: 115806540-115806618) in endometrial cancer was 68.3% (41/60), the specificity in benign samples was 80% (32/40), and the ROC area was 0.767;
the positive rate of CELF4 (Chr 18: 37243195-37243249) in endometrial cancer was 61.7% (37/60), the specificity in benign samples was 90% (36/40), and the ROC area was 0.776;
the positive rate of CELF4 (Chr 18: 37244217-37244299) in endometrial cancer was 73.3% (44/60), the specificity in benign samples was 87.5% (35/40), and the ROC area was 0.847;
the positivity rate of CELF4 (Chr 18: 37244683-37244784) in endometrial cancer was 86.7% (52/60), the specificity in benign samples was 62.5% (25/40), and the ROC area was 0.792;
the positive rate of HAND2 (Chr 4: 173526387-173526445) in endometrial cancer was 46.7% (28/60), the specificity in benign samples was 100% (40/40), and the ROC area was 0.742;
the positive rate of HAND2 (Chr 4: 173527003-173527077) in endometrial cancer is 60% (36/60), the specificity in benign samples is 95% (38/40), and the ROC area is 0.811;
the positive rate of HAND2 (Chr 4: 173527734-173527835) in endometrial cancer was 78.3% (47/60), the specificity in benign samples was 90% (36/40), and the ROC area was 0.882;
the positive rate of HS3ST2 (Chr 16: 22813832-22813912) in endometrial cancer is 86.7% (52/60), the specificity in benign samples is 95% (38/40), and the ROC area is 0.915;
the positive rate of HS3ST2 (Chr 16: 22814061-22814120) in endometrial cancer is 66.7% (40/60), the specificity in benign samples is 95% (38/40), and the ROC area is 0.790;
the positive rate of HS3ST2 (Chr 16: 22814470-22814540) in endometrial cancer was 91.7% (55/60), the specificity in benign samples was 65% (26/40), and the ROC area was 0.797.
Through the comparative analysis of CDO1, CELF4, HAND2 and HS3ST2 multi-methylation regions, the methylation region selected by the CDO1 gene is Chr5:115806540 and 115806618, the methylation region selected by the CELF4 gene is Chr18:37244217-37244299, the methylation region selected by HAND2 gene is Chr4:173527734-173527835, HS3ST2 were selected to have a methylation region of Chr16: 22814061-22814120).
The joint detection based on the optimal methylation regions of CDO1, CELF4, HAND2 and HS3ST2 can involve two genes in interpretation, or three or four genes simultaneously in interpretation analysis, and the following table results can be obtained:
TABLE 1 Combined detection Performance of two genes
TABLE 2 Combined detection Performance of three genes
TABLE 3 Combined detection of Performance of four genes
From the above table we can see that any two of the 4 genes were positive at the same time, with a positive rate of 98.3% in endometrial cancer (59/60), specificity of 100% in benign samples (40/40) and a ROC area of 0.990.
Therefore, the combined detection of CDO1, CELF4, HAND2 and HS3ST2 has the highest detection rate of endometrial cancer and good specificity as can be analyzed by example 1. Preliminary application of DNA methylation to early detection of endometrial cancer was confirmed by validation of small samples, and high detection rates and low false positive rates could be achieved by cervical exfoliated cells.
Example 2
The other components in the kit are the same as in example 1. In example 1, a small amount of single-center samples were tested, and then nearly 1000 samples of multi-center samples were collected, including Beijing collaborating hospital, Beijing university International Hospital, general Hospital of the liberation military, Nemontage autonomous region people Hospital, Hebei Cangzhou City center Hospital, 450 samples of endometrial cancer, 650 samples of benign samples of endometrium, and 1100 samples in total.
Compared with histopathological pathological results, the combined ROC curve area obtained by using the methylation detection kit is 0.98, the overall specificity is 98.5 percent (640/650), and the detection sensitivity of endometrial cancer is 98.9 percent (445/450).
The kit provided by the invention is verified by a small amount of samples to be a large amount of sample researches, and the DNA methylation has high accuracy on early detection of endometrial cancer and can be detected by cervical exfoliated cells. The invention applies a special primer probe design method and a sample pretreatment kit independently developed by a company, realizes multi-gene joint detection and function complementation, and obviously improves the detection of early endometrial cancer.
The inventive concept is explained in detail herein using specific examples, which are given only to aid in understanding the core concepts of the invention. It should be understood that any obvious modifications, equivalents and other improvements made by those skilled in the art without departing from the spirit of the present invention are included in the scope of the present invention.
Sequence listing
<110> Beijing-originated Poa-gathering Biotech Co., Ltd
<120> endometrial cancer early screening diagnosis marker, primer probe and kit
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aatttcacca gagagggggt gaaattgc 28
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tataattagc gtcgcgaatt ata 23
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aaccaagttt aaagtgattg gttcg 25
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<213> Artificial Sequence (Artificial Sequence)
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cttccacctg gttattttag gtggaaggc 29
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acccttagtc gcggggttgg t 21
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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catctatttc gggcgcggag atgcgg 26
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aatccaatgc gcgttcggat tttcg 25
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<213> Artificial Sequence (Artificial Sequence)
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attccatgta tataaagatg gttacg 26
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ctaattcaat tgatattgaa ttaggc 26
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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atctataacg ggttcggtag tagtt 25
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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cccaaaataa gattgggttt tgggcg 26
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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taaactttcg taggggtaga aagtttagc 29
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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cttctagcgg gcgtcgatga agaga 25
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<213> Artificial Sequence (Artificial Sequence)
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attataaaaa ataatagtat ttataatcg 29
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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actcaataag ataaatttta ttgagtgc 28
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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tcctctgttc gttcggagga ttta 24
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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acaataaaat taaaaagttt tattgtcg 28
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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caaaatttcg ttgatgtaga aattttggc 29
<210> 24
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
aaaagttcga ggcgtcgttt ttgt 24
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<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ctattaatgg atttagagta ttaatagcg 29
<210> 26
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
aaaccaatat attgattatt ggtttgc 27
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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caatttcgtc gaattgcgcg 20
<210> 28
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
caaacaccca ttagggtagg gtgtttgcg 29
<210> 29
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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accaccccga tttgtggggg tggtgc 26
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<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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aaattcggcg cgatttcgat ttgga 25
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
cccccactgt aagagagtgg gggcg 25
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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aatccaatta aattttttat tggattgc 28
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
cccccggcgc gggttcgggg gatt 24
<210> 34
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
aaaactactt ttggttagta gttttcg 27
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<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
atcccaattt tgatgatatt gggatgc 27
<210> 36
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
aaaaagcgtt acgcgagttt tttag 25
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<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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aggttaaata tagttgttga 20
<210> 38
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
caacccaaac ccccaac 17
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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tagttggggg tttgggtt 18
Claims (10)
1. The marker for early screening and diagnosing endometrial cancer is characterized by comprising at least one methylation region in any one of the following genes selected from target genes CDO1, CELF4, HAND2 and HS3ST 2:
CDO1 gene: chr5:115805377-115805492, Chr5:115806258-115806356, Chr5: 115806540-115806618;
CELF4 gene: chr18:37243195-37243249, Chr18:37244217-37244299, Chr18: 37244683-37244784;
HAND2 gene: chr4:173526387-173526445, Chr4:173527003-173527077, Chr4: 173527734-173527835;
HS3ST2 gene: chr16:22813832-22813912, Chr16: 22814061-22814120, and Chr16: 22814470-22814540.
2. The marker for early screening diagnosis of endometrial cancer according to claim 1, wherein the following methylation regions are selected from each of the target genes CDO1, CELF4, HAND2 and HS3ST 2:
CDO1 gene: chr5:115806540 and 115806618;
CELF4 gene: chr18: 37244217-37244299;
HAND2 gene: chr4: 173527734-173527835;
HS3ST2 gene: chr16: 22813832-22813912.
3. The detection primer for early screening and diagnosing the endometrial cancer is used for correspondingly detecting the methylation state of the methylation region of the marker gene in claim 1, the primer is designed to adopt a clamp ring type structure, a sequence which is 5-10bp long and is complementarily paired with a 3 ' end is added to the 5 ' end of a primer nucleotide sequence, the sequence does not contain CG sites, the last two bases at the tail end of the 3 ' end are CG, and the Tm value of a double-stranded binding region of the primer is consistent with the annealing temperature in a PCR reaction system.
4. The detection primer for early screening and diagnosing endometrial cancer according to claim 3, wherein the nucleotide sequence of the detection primer is as follows:
CDO1 gene:
the detection primers corresponding to Chr5:115805377-115805492 are SEQ ID NO 1-2,
the detection primers corresponding to Chr5:115806258 and 115806356 are SEQ ID NO. 4-5,
the detection primers corresponding to Chr5:115806540 and 115806618 are SEQ ID NO 7-8;
CELF4 gene:
the detection primers corresponding to Chr18:37243195 and 37243249 are SEQ ID NO 10-11,
the detection primers corresponding to Chr18:37244217-37244299 are SEQ ID NO 13-14,
the detection primers corresponding to Chr18:37244683 and 37244784 are SEQ ID NO 16-17;
among the HAND2 genes:
the corresponding detection primers of Chr4:173526387 and 173526445 are SEQ ID NO. 19-20,
the corresponding detection primers of Chr4:173527003-173527077 are SEQ ID NO. 22-23,
the corresponding detection primers of Chr4:173527734-173527835 are SEQ ID NO. 25-26;
HS3ST2 gene:
the corresponding detection primers of Chr16:22813832 and 22813912 are SEQ ID NO 28-29,
the corresponding detection primers of Chr16: 22814061 and 22814120 are SEQ ID NO. 31-32,
the corresponding detection primers of Chr16: 22814470 and 22814540 are SEQ ID NO 34-35.
5. The detection probe for early screening and diagnosing the endometrial cancer is characterized by being used for correspondingly detecting the marker gene in claim 1, the nucleotide sequence of the detection probe is designed to adopt a snap ring type structure, a section of sequence which is 3-6bp long and is complementarily paired with a 3-4bp region close to the tail end of a 3 'end is added at the 5' end of the nucleotide sequence, the section of sequence does not contain CG sites, the Tm value of a double-stranded binding region of the probe is higher than the annealing temperature in a PCR reaction system by 2-15 ℃, and the binding free energy of the nucleotide sequence of the probe and the marker gene sequence is higher than the snap ring structure formed by the probe;
the 5 'end of the detection probe is marked with a fluorescent group, the 3' end of the detection probe is marked with a quenching group, and different marker gene detection probes in the same detection system are marked with different fluorescent groups.
6. The detection probe for early screening and diagnosing endometrial cancer according to claim 5, wherein the nucleotide sequence of the detection probe is as follows:
CDO1 gene detection probe: 9, SEQ ID NO;
CELF4 gene detection probe: 15, SEQ ID NO;
HAND2 gene detection probe: 27, SEQ ID NO;
HS3ST2 gene detection probe: SEQ ID NO 30.
7. The detection kit for early screening and diagnosing endometrial cancer, which is characterized by comprising the detection primer as defined in claim 3 or 4 and the detection probe as defined in claim 5 or 6.
8. The detection kit for early screening and diagnosing endometrial cancer according to claim 7, further comprising an internal reference gene detection primer and a probe, wherein the internal reference gene is GAPDH, the nucleotide sequence of the internal reference gene detection primer is shown in SEQ ID NO: 37-38, the nucleotide sequence of the internal reference gene detection probe is shown in SEQ ID NO:39, the 5 'end of the nucleotide sequence of the internal reference gene detection probe is labeled with a fluorescent group, the 3' end of the nucleotide sequence of the internal reference gene detection probe is labeled with a quenching group, and the fluorescent group labeled by the internal reference gene detection probe and the fluorescent group labeled by the marker gene detection probe in the same detection system are different.
9. The detection kit for early screening and diagnosing endometrial cancer according to claim 7, further comprising a PCR reaction solution and a sample methylation pretreatment reagent; the sample is a cervical exfoliated cell, and the sample methylation pretreatment reagent comprises a cell genome DNA extraction reagent and a DNA bisulfite conversion reagent.
10. The detection kit for early screening and diagnosing endometrial cancer according to claim 9, wherein the PCR reaction solution comprises, per one person, 0.5 to 1 μ L of Taq DNA polymerase with methylation characteristics at a concentration of 1U/μ L, 2 to 5 μ L of dNTPs with a concentration of 10 mM, and2 to 5 mM of Mg2+2-6 μ L, 10 XDNA polymerase buffer 5 μ L and purified water to make up 15 μ L.
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
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CN202110898883.2A CN113337614A (en) | 2021-08-05 | 2021-08-05 | Marker, primer probe and kit for early screening and diagnosis of endometrial cancer |
CN202111228571.7A CN113755603B (en) | 2021-08-05 | 2021-10-21 | Marker, primer probe and kit for early screening and diagnosis of endometrial cancer |
EP22155652.5A EP4130297A1 (en) | 2021-08-05 | 2022-02-08 | Markers, primers, probes and kit for early screening and diagnosis of endometrial cancer |
PCT/CN2022/095439 WO2023010964A1 (en) | 2021-08-05 | 2022-05-27 | Marker for early screening and diagnosis of endometrial cancer, primer probe and reagent test kit |
US17/883,872 US20230076141A1 (en) | 2021-08-05 | 2022-08-09 | Markers, primers, probes and kit for early screening and diagnosis of endometrial cancer |
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CN202110898883.2A CN113337614A (en) | 2021-08-05 | 2021-08-05 | Marker, primer probe and kit for early screening and diagnosis of endometrial cancer |
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CN202111228571.7A Active CN113755603B (en) | 2021-08-05 | 2021-10-21 | Marker, primer probe and kit for early screening and diagnosis of endometrial cancer |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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CN113755603A (en) * | 2021-08-05 | 2021-12-07 | 北京起源聚禾生物科技有限公司 | Marker, primer probe and kit for early screening and diagnosis of endometrial cancer |
CN114480655A (en) * | 2022-03-03 | 2022-05-13 | 北京起源聚禾生物科技有限公司 | DNA methylation marker combination and application thereof, ovarian cancer early detection primer probe and kit |
CN114645094A (en) * | 2022-03-21 | 2022-06-21 | 武汉凯德维斯生物技术有限公司 | Endometrial cancer biomarker and application thereof |
WO2023010964A1 (en) * | 2021-08-05 | 2023-02-09 | 北京起源聚禾生物科技有限公司 | Marker for early screening and diagnosis of endometrial cancer, primer probe and reagent test kit |
CN116144757A (en) * | 2023-01-19 | 2023-05-23 | 广州市妇女儿童医疗中心 | Application of methylation site as endometriosis molecular diagnostic marker |
CN116356029A (en) * | 2023-03-29 | 2023-06-30 | 北京优迅医疗器械有限公司 | Application of TXNRD1 gene methylation level detection in endometrial cancer diagnosis and/or screening |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
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EP4265738A1 (en) | 2022-03-03 | 2023-10-25 | Beijing OriginPoly Bio-Tec Co., Ltd. | Dna methylation marker combination, use, primer probe for early detection of ovarian cancer and kit |
CN116064797B (en) * | 2022-08-29 | 2023-10-20 | 广州达健生物科技有限公司 | Endometrial cancer gene methylation level detection reagent and application thereof |
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CN110387420A (en) * | 2019-08-26 | 2019-10-29 | 武汉艾米森生命科技有限公司 | Kit and application for carcinoma of endometrium diagnosis |
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CN113337614A (en) * | 2021-08-05 | 2021-09-03 | 北京起源聚禾生物科技有限公司 | Marker, primer probe and kit for early screening and diagnosis of endometrial cancer |
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- 2021-08-05 CN CN202110898883.2A patent/CN113337614A/en not_active Withdrawn
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CN109069670A (en) * | 2016-07-29 | 2018-12-21 | 台北医学大学 | The diagnostic method of gynecological tumor |
CN110387420A (en) * | 2019-08-26 | 2019-10-29 | 武汉艾米森生命科技有限公司 | Kit and application for carcinoma of endometrium diagnosis |
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Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
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CN113755603A (en) * | 2021-08-05 | 2021-12-07 | 北京起源聚禾生物科技有限公司 | Marker, primer probe and kit for early screening and diagnosis of endometrial cancer |
WO2023010964A1 (en) * | 2021-08-05 | 2023-02-09 | 北京起源聚禾生物科技有限公司 | Marker for early screening and diagnosis of endometrial cancer, primer probe and reagent test kit |
CN114480655A (en) * | 2022-03-03 | 2022-05-13 | 北京起源聚禾生物科技有限公司 | DNA methylation marker combination and application thereof, ovarian cancer early detection primer probe and kit |
CN114645094A (en) * | 2022-03-21 | 2022-06-21 | 武汉凯德维斯生物技术有限公司 | Endometrial cancer biomarker and application thereof |
CN114645094B (en) * | 2022-03-21 | 2023-10-20 | 武汉凯德维斯生物技术有限公司 | Biomarker for endometrial cancer and application thereof |
CN116144757A (en) * | 2023-01-19 | 2023-05-23 | 广州市妇女儿童医疗中心 | Application of methylation site as endometriosis molecular diagnostic marker |
CN116144757B (en) * | 2023-01-19 | 2023-10-03 | 广州市妇女儿童医疗中心 | Application of methylation site as endometriosis molecular diagnostic marker |
CN116356029A (en) * | 2023-03-29 | 2023-06-30 | 北京优迅医疗器械有限公司 | Application of TXNRD1 gene methylation level detection in endometrial cancer diagnosis and/or screening |
CN116356029B (en) * | 2023-03-29 | 2023-12-12 | 北京优迅医疗器械有限公司 | Application of TXNRD1 gene methylation level detection in endometrial cancer diagnosis and/or screening |
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CN113755603B (en) | 2022-05-20 |
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