CN114480661A - Endometrium benign and malignant lesion combined marker, detection primer probe set and kit - Google Patents

Endometrium benign and malignant lesion combined marker, detection primer probe set and kit Download PDF

Info

Publication number
CN114480661A
CN114480661A CN202210401707.8A CN202210401707A CN114480661A CN 114480661 A CN114480661 A CN 114480661A CN 202210401707 A CN202210401707 A CN 202210401707A CN 114480661 A CN114480661 A CN 114480661A
Authority
CN
China
Prior art keywords
seq
gene
methylation
detection
region
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210401707.8A
Other languages
Chinese (zh)
Inventor
王林海
刘沛
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Originpoly Bio Tec Co Ltd
Original Assignee
Beijing Originpoly Bio Tec Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Originpoly Bio Tec Co Ltd filed Critical Beijing Originpoly Bio Tec Co Ltd
Priority to CN202210401707.8A priority Critical patent/CN114480661A/en
Publication of CN114480661A publication Critical patent/CN114480661A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a combined marker, a detection primer probe set and a kit for benign and malignant lesions of endometrium, wherein the combined marker comprises specific methylation regions of 26 target genes, detection primers and probes aiming at the methylation regions can be used for detecting through fluorescence quantitative PCR, and the detection result can be used for evaluating the risk of benign and malignant lesions of endometrium by adopting a logistic regression algorithm, and comprises the steps of effectively distinguishing benign and malignant lesions such as hysteromyoma, uterine sarcoma, atypical hyperplasia of endometrium and endometrial carcinoma. The invention significantly improves the sensitivity and specificity of the detection of the malignant lesions of the endometrium by the combined detection of multiple gene methylation. The kit is particularly suitable for differential diagnosis of benign and malignant endometrial lesions by taking cervical exfoliated cells as samples, and has the advantages of simple and convenient sampling, high detection speed, high accuracy, high specificity and the like.

Description

Endometrium benign and malignant lesion combined marker, detection primer probe set and kit
Technical Field
The invention relates to a endometrium benign and malignant lesion combined detection marker, a detection primer probe set and a kit.
Background
Uterine fibroids are the most common benign tumors in female genitalia, and are also called fibroids and uterine fibroids. The incidence increases with age, and is often seen in women of 30-55 years old. Uterine fibroids can be degenerated, mostly benign, including vitreoid degeneration, cystic degeneration, red degeneration, and a few malignant, such as sarcoma, with rapid increase in the malignant time in the short term accompanied by vaginal bleeding, and after menopause, they do not shrink but grow larger, and are basically malignant. Some malignant changes have no symptoms and are easy to be ignored.
Since the mechanisms of occurrence and development of uterine sarcoma and endometrial cancer are different, there is a need for an objective, reproducible, and highly specific detection method to better distinguish the possibility of benign and malignant lesions of the endometrium. At present, effective means for identifying benign and malignant endometrial lesions are lacking. At present, fourteen-five key items of birth health and women and children health guarantee are special, and the exploration of the difference of molecular mechanisms between hysteromyoma and uterine sarcoma is key.
Chinese patent publication No. CN113755603 discloses that early-stage endometrial cancer is screened by methylation detection through combination of four target genes, namely COD1, CELF4, HAND2 and HS3ST 2.
Currently, there is no method capable of non-invasive detection of multiple uterine pathologies such as uterine fibroids, uterine sarcoma, endometrial cancer, etc. simultaneously.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a combined marker for benign and malignant lesions of endometrium, a detection primer probe set and a kit, which can accurately and quickly distinguish different benign and malignant lesions of endometrium, can use cervical exfoliated cells as a detection sample, cannot damage a patient, and is easier to accept by the patient.
The technical scheme of the invention is detailed as follows:
in a first aspect, the invention provides a combined endometrial benign and malignant lesion marker comprising 26 methylated regions having the nucleotide sequence shown in SEQ ID NO:82-107, comprising ADCY4, ADRA1A, AMPD3, CDO1, CELF4, CHAD, CYP26C1, DRD4, FZD7, GNA12, GRM6, GYPC, HAND2, HSPA1L, HS3ST2, ITGA5, LTB4R2, MAGI2, MTMR7, NDS 2, NEFM, PTGDR, RASSF1, SPDYA, TJP2, TRH totaling 26 genes of interest.
In a second aspect, the invention also provides a primer probe set for joint detection of benign and malignant endometrial lesions, which comprises a nucleotide sequence shown in SEQ ID NO. 1-78 and is used for correspondingly detecting the methylation states of the methylation regions of the 26 target genes.
Optionally or preferably, the primer probe set further comprises a detection primer and a probe of an internal reference gene GAPDH, and the nucleotide sequence is shown as SEQ ID NO: 79-81.
The 5 'end of the nucleotide sequence of the detection probe of each gene is provided with a fluorescent group, and the fluorescent groups at the 5' ends of the detection probes of different genes are different in the same detection system. The fluorophore can be FAM, ROX, CY5, HEX, JOE, etc.
In a third aspect, the invention provides a combined detection kit for benign and malignant endometrial lesions, which comprises the primer probe set.
Alternatively or preferably, the kit may further comprise a PCR reaction solution and a sample methylation pretreatment reagent.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention screens 26 related marker genes, determines the optimal methylation regions thereof, and can effectively distinguish hysteromyoma, uterine sarcoma and endometrial carcinoma by integrating the state detection of the methylation regions. Due to the variety of benign and malignant endometrial lesions, a multi-gene methylation region is selected for combined detection, so that functional complementation is formed, the detection sensitivity is obviously improved, and the kit has high specificity on normal and benign endometrial tumors. The combined marker can detect 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 primer probe designed aiming at the methylation region of the target gene obviously improves the sensitivity and specificity of detection, increases the accuracy of detection and reduces the detection error. 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, multiple 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, 26 gene methylation sites are accurately detected, 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 totally-closed form, the operation is simpler and more convenient, and the possibility of cross contamination is avoided.
3. 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 PCR reaction solution and the sample methylation pretreatment reagent in the kit adopt reagents with specific components and proportions, and the sample methylation pretreatment reagent (Beijing-originated Poa Biotech Co., Ltd., Beijing mechanical preparation No. 20210020) comprises a cell genome DNA extraction reagent and a DNA bisulfite conversion reagent.
The DNA extraction is carried out on the exfoliated cervical cells, the genome DNA extraction reagent of the cells is greatly improved in both the yield of the DNA and the purity of the DNA by contrast screening in the development process, 2mL of cervical exfoliated cell preservative fluid is extracted, the total amount of the DNA is between 4 and 8 mu g, the OD260/280 is between 1.8 and 2.0, and better yield and purity are kept. The main indicators for the comparison of the transformation process are two, namely the transformation rate of bisulfite, how many C in the sequence can be transformed into U, and the purification efficiency after transformation, namely the final yield of bisDNA. When the specific 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.
The PCR reaction solution contains Taq polymerase with methylation specificity, and 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.
In conclusion, the invention detects the multiple gene methylation states of the uterine fibroid, the uterine sarcoma and the endometrial cancer by a multiple gene methylation combined detection mode, further identifies the benign and malignant conditions of the endometrium, has high detection accuracy, has very good detection rate for a template with low concentration, short detection time, no wound to a patient and better patient compliance.
Drawings
FIG. 1 shows the ROC curves for the combined detection of methylation regions of 26 target genes, the left graph shows the ROC curve for uterine sarcoma, and the right graph shows the ROC curve for endometrial carcinoma.
Detailed Description
The technical solutions of the present invention are explained and illustrated in detail below with reference to preferred embodiments and drawings so that those skilled in the art can better understand the present invention and implement it.
The reagents, instruments and experimental method steps described in the examples, unless otherwise specified, are conventional in the art and are readily available to those of ordinary skill in the art.
Example 1
Screening of the Illumina Infinium Methylation EPIC BeadChip chip (containing 853,307 CpG sites) Methylation genes was performed by selecting 20 uterine fibroids, 20 uterine sarcomas and 20 endometrial carcinoma paraffin-embedded tissue samples.
Comparing and analyzing the detection results of the benign and malignant endometrial samples of different types by a significant methylation differential locus algorithm, and screening out 26 genes which are shared by the first 100 th sites of deltaBeta (the methylation level of the benign endometrial control sample-the positive endometrial methylation level) as gene markers, wherein the genes are respectively as follows: ADCY4, ADRA1A, AMPD3, CDO1, CELF4, CHAD, CYP26C1, DRD4, FZD7, GNA12, GRM6, GYPC, HAND2, HSPA1L, HS3ST2, ITGA5, LTB4R2, MAGI2, MTMR7, ndifs 2, NEFM, PTGDR, RASSF1, SPDYA, TJP2, TRH. Methylation specific PCR amplification verification was then performed. Since the CpG regions in each gene are distributed in a large number, the gene needs to be transferred into RT-PCR for verification and analysis of the specific methylation position of each gene. Finally, a methylation region fragment was identified in each gene of the above gene markers.
And (3) designing and screening a primer probe aiming at the determined methylation region fragment to finally obtain the nucleotide sequence of SEQ ID NO 1-78.
Example 2
Detection tests for detecting endometrial benign and malignant lesion related genes ADCY4, ADRA1A, AMPD3, CDO1, CELF4, CHAD, CYP26C1, DRD4, FZD7, GNA12, GRM6, GYPC, HAND2, HSPA1L, HS3ST2, ITGA5, LTB4R2, MAGI2, MTMR7, NDS 2, NEFM, GDPTR, RAUFSSF 1, SPDYA, TJP2, TRH methylation kit.
The specific nucleotide sequences of the primers and probes used are shown in the following table:
ADCY4-F1 GGGAGGGGGTCGCG SEQ ID NO:1
ADCY4-R1 GAAATCACGCTCGCCGCTACG SEQ ID NO:2
ADCY4-FP1 FAM-GCGTTTGTCGTTCGGG-BHQ1 SEQ ID NO:3
ADRA1A-F1 TAGGTAGTTTTCGGGATCG SEQ ID NO:4
ADRA1A-R1 GACGCACTCACCTAAAACGCCG SEQ ID NO:5
ADRA1A-FP1 FAM-TTTCGCGCGCGAGTT-BHQ1 SEQ ID NO:6
AMPD3-F1 GTTGGGAAAAGTTGTGCG SEQ ID NO:7
AMPD3-R1 CCGCCGAACTTCCACACCG SEQ ID NO:8
AMPD3-FP1 FAM-GACGTCGGGTAAGTTCGGGC-BHQ1 SEQ ID NO:9
CDO1-F1 ataacGTTTATATTTTTAAGTTATCG SEQ ID NO:10
CDO1-R1 gattagACCCTCTACTAATCCG SEQ ID NO:11
CDO1-FP1 FAM-catctATTTCGGGCGCGGAGATGCGG-BHQ1 SEQ ID NO:12
CELF4-F1 attccatGTATATAAAGATGGTTACG SEQ ID NO:13
CELF4-R1 gattaagAACTATAACTTAATCCG SEQ ID NO:14
CELF4-FP1 ROX-atctaTAACGGGTTCGGTAGTAGTT-BHQ2 SEQ ID NO:15
CHAD-F1 TCGAGTTTGAAGTGGGAGATCG SEQ ID NO:16
CHAD-R1 ACTACCTACGCTTTAAAAAAACG SEQ ID NO:17
CHAD-FP1 ROX-ACGTCGGGGCGGTTTTTACG-BHQ2 SEQ ID NO:18
CYP26C1-F1 GTTAGGAGTATTGCGGGGCG SEQ ID NO:19
CYP26C1-R1 ACTTCCCCGCTCTTCGCAAACGC SEQ ID NO:20
CYP26C1-FP1 CY5-TTGCGCGTTGGGTTCGTTT-BHQ2 SEQ ID NO:21
DRD4-F1 GAGGGAATGGAGGAGGGAGCG SEQ ID NO:22
DRD4-R1 TACACAATTAATCCTCGCGTAACCG SEQ ID NO:23
DRD4-FP1 ROX-GTCGATCGTTTAGTTGTTCGTTT-BHQ2 SEQ ID NO:24
FZD7-F1 ATTTTTAGAATTAGTTTTTTTAACG SEQ ID NO:25
FZD7-R1 AAAACCCGAACTCAAACCACC SEQ ID NO:26
FZD7-FP1 FAM-GTCGTGCGCGCGGGCGGGG-BHQ1 SEQ ID NO:27
GNA12-F1 TTGAAAGAAAGAATTTTAGTTTGGGCG SEQ ID NO:28
GNA12-R1 TCGCCAAAAAAAACGAACATCCC SEQ ID NO:29
GNA12-FP1 ROX-TTTCGGCGTCGATTTCGTTTCGA-BHQ2 SEQ ID NO:30
GRM6-F1 AGTGGTCGGGCGGGATTTCGGGCG SEQ ID NO:31
GRM6-R1 AATTAACTCAACCGACCTACCCG SEQ ID NO:32
GRM6-FP1 JOE-GTTCGGCGTTTGTAAGAGCGAGACG-BHQ1 SEQ ID NO:33
GYPC-F1 GTGATTTAGGTGTCGTTTTTTTTCG SEQ ID NO:34
GYPC-R1 AACTAACCAAACCGAACCAAACCG SEQ ID NO:35
GYPC-FP1 ROX-AGGAGTTCGGGAGCGCGATTT-BHQ2 SEQ ID NO:36
HAND2-F1 ctattaatGGATTTAGAGTATTAATAGCG SEQ ID NO:37
HAND2-R1 tttggttATATAACTAATAACCAAACG SEQ ID NO:38
HAND2-FP1 CY5-caatTTCGTCGAATTGCGCG-BHQ2 SEQ ID NO:39
HSPA1L-F1 TAGTGTTTTATGTTCGATTGTATT SEQ ID NO:40
HSPA1L-R1 CGCTAAACCCGCAAAACACCG SEQ ID NO:41
HSPA1L-FP1 HEX-TCGGGTCGTCGAATTTGCGG-BHQ1 SEQ ID NO:42
HS3ST2-F1 caaacacccATTAGGGTAGGGTGTTTGCG SEQ ID NO:43
HS3ST2-R1 tggtggggCTAAACACCCCCACCACG SEQ ID NO:44
HS3ST2-FP1 HEX-aaatTCGGCGCGATTTCGATTTGGA-BHQ1 SEQ ID NO:45
ITGA5-F1 GGCGTTATGGGGAGTCGGAC SEQ ID NO:46
ITGA5-R1 ACCCCCGACCCTAAATAACG SEQ ID NO:47
ITGA5-FP1 CY5-TTCGGCGTCGATTTTCGTT-BHQ2 SEQ ID NO:48
LTB4R2-F1 GGCGGGTTGGCGGTTTGTAC SEQ ID NO:49
LTB4R2-R1 AAAAACCACAAAAAACGACGT SEQ ID NO:50
LTB4R2-FP1 CY5-GGCGTTGGTCGACGGCGCGGT-BHQ2 SEQ ID NO:51
MAGI2-F1 TTTCGTTTTAATTTTTTTTTACG SEQ ID NO:52
MAGI2-R1 TCCGATAAAACAAAAATAACGA SEQ ID NO:53
MAGI2-FP1 JOE-ATTTCGCGTTAGGACGTTCGTA-BHQ1 SEQ ID NO:54
MTMR7-F1 AGAGAGAGCGAGTAGTTTTCG SEQ ID NO:55
MTMR7-R1 GACCCTAAAAAACCCACCTCC SEQ ID NO:56
MTMR7-FP1 HEX-GGCGGCGGCGTAGGTG-BHQ1 SEQ ID NO:57
NDUFS2-F1 TTTGTTTTAGTTTTAGTAGTAGCG SEQ ID NO:58
NDUFS2-R1 ATCGTATTTCCAAAAAAACTCAACG SEQ ID NO:59
NDUFS2-FP1 HEX-GGCGTTCGAGTTAGGTAGGACGT-BHQ1 SEQ ID NO:60
NEFM-F1 GGTCGTCGTTTTTACGGGGC SEQ ID NO:61
NEFM-R1 CTATCACAACGTTCTACCGACC SEQ ID NO:62
NEFM-FP1 HEX-TAGCGTCGCGGTTATAAAT-BHQ1 SEQ ID NO:63
PTGDR-F1 TTGGTTATTTTTTTTTTGTCGTTTC SEQ ID NO:64
PTGDR-R1 CTAACGCTAACATTAAAAAACG SEQ ID NO:65
PTGDR-FP1 FAM-AGCGCGTTAGGGTCGAGT-BHQ1 SEQ ID NO:66
RASSF1-F1 GGAGTTTGAGTTTATTGAGTTGC SEQ ID NO:67
RASSF1-R1 CGTTAACACGCTCCAACCGAATA SEQ ID NO:68
RASSF1-FP1 ROX-TTCGTTGGGCGCGTTGGGA-BHQ2 SEQ ID NO:69
SPDYA-F1 TTTGAGTAATTAGGGTAATTACG SEQ ID NO:70
SPDYA-R1 GATTAAACAACCAAACCCCCGAAA SEQ ID NO:71
SPDYA-FP1 CY5-TTCGCGTGTGTCGGGCGGGGA-BHQ2 SEQ ID NO:72
TJP2-F1 GCGAGTAGTTGCGTTTTTTTTTC SEQ ID NO:73
TJP2-R1 CTATCACCTACTTCCTTAAAACCG SEQ ID NO:74
TJP2-FP1 CY5-TCGGGGTTCGCGCGTTTAGA-BHQ2 SEQ ID NO:75
TRH-F1 ATTTTTAGAGGTGGTGTAGAAAC SEQ ID NO:76
TRH-R1 GAAAACGATAAAAACCGAAACCG SEQ ID NO:77
TRH-FP1 JOE-TTCGCGTCGGCGTTTTATTTTG-BHQ1 SEQ ID NO:78
GAPDH-F AGGTTAAATATAGTTGTTGA SEQ ID NO:79
GAPDH-R CAACCCAAACCCCCAAC SEQ ID NO:80
GAPDH-FP JOE-TAGTTGGGGGTTTGGGTT-BHQ1 SEQ ID NO:81
note: in this table, F represents the forward detection primer, R represents the reverse detection primer, FP represents the detection probe; 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:
Figure DEST_PATH_IMAGE002
selecting 40 examples of hysteromyoma samples with known and definite pathological information results: 40 cases of uterine sarcoma samples, 40 cases of endometrial carcinoma samples. 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 kit (nucleic acid extraction or purification reagent, Beijing original Poa biological technology Co., Ltd., Beijing Dajiu Tokyo et al 20210020) was used as a cell genome DNA extraction reagent to extract genome DNA from 120 samples of good and malignant endometrium cervical exfoliated cells and to monitor DNA quality. The total amount of DNA is between 4 mug and 8 mug, OD260/280 is between 1.8 and 2.0, and good yield and purity are kept.
2. A bisulfite conversion kit (methylation detection sample pretreatment kit, Beijing-originated Poa Biotech Co., Ltd., Beijing Dai mechanical Co., Ltd., No. 20200110) is adopted as a DNA bisulfite conversion reagent to carry out bisulfite conversion on the extracted DNA, wherein unmethylated cytosine (C) in the DNA is converted into uracil (U), and the methylated cytosine (C) is not changed, so that 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) 1
dNTPs（10mM） 4
Mg2+（2-5mM） 5
10 XDNA polymerase buffer 1.5
Purified water Make up to 15. mu.L
Primer Probe Mixed solution 1 (5. mu.L/person)
Components One part addition amount (mu L)
ADCY4/CHAD/CYP26C1/HSPA1L/MAGI2-F（100μM） 0.02-0.08
ADCY4/CHAD/CYP26C1/HSPA1L/MAGI2-R（100μM） 0.02-0.08
ADCY4/CHAD/CYP26C1/HSPA1L/MAGI2-FP（100μM） 0.02-0.04
Purified water Make up to 5. mu.L
Primer probe mixed solution 2 (5 mu L/person)
Components One part addition amount (mu L)
ADRA1A/GYPC/TJP2/NEFM/GRM6-F（100μM） 0.02-0.08
ADRA1A/GYPC/TJP2/NEFM/GRM6-R（100μM） 0.02-0.08
ADRA1A/GYPC/TJP2/NEFM/GRM6-FP（100μM） 0.02-0.04
Purified water Make up to 5. mu.L
Primer Probe Mixed solution 3 (5. mu.L/person)
Components One part addition amount (mu L)
AMPD3/DRD4/ITGA5/NDUFS2/TRH-F（100μM） 0.02-0.08
AMPD3/DRD4/ITGA5/NDUFS2/TRH-R（100μM） 0.02-0.08
AMPD3/DRD4/ITGA5/NDUFS2/TRH-FP（100μM） 0.02-0.04
Purified water Make up to 5. mu.L
Primer Probe Mixed solution 4 (5. mu.L/person)
Components One part addition amount (mu L)
FZD7/GNA12/LTB4R2/MTMR7-F（100μM） 0.02-0.08
FZD7/GNA12/LTB4R2/MTMR7-R（100μM） 0.02-0.08
FZD7/GNA12/LTB4R2/MTMR7-FP（100μM） 0.02-0.04
Purified water Make up to 5. mu.L
Primer Probe Mixed solution 5 (5. mu.L/person)
Components One part addition amount (mu L)
PTGDR/RASSF1/SPDYA-F（100μM） 0.02-0.08
PTGDR/RASSF1/SPDYA-R（100μM） 0.02-0.08
PTGDR/RASSF1/SPDYA-FP（100μM） 0.02-0.04
Purified water Make up to 5. mu.L
Primer Probe Mixed solution 6 (5. mu.L/person)
Components One part addition amount (mu L)
CDO1/CELF4/HAND2/HS3ST2-F（100μM） 0.02-0.08
CDO1/CELF4/HAND2/HS3ST2-R（100μM） 0.02-0.08
CDO1/CELF4/HAND2/HS3ST2-FP（100μM） 0.02-0.04
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
And respectively adding 5 mu L of negative and positive quality control products and the transformed Bis-DNA clinical samples into the prepared system, namely performing PCR reaction on each sample by using six groups of primer probe mixed liquor, wherein each reaction system is provided with PCR reaction liquid. The conditions under which the PCR reaction was carried out were: 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 (reference gene GAPDH) 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) calculate Δ Ct value: Δ Ct (target gene) = Ct (target gene) -Ct (reference gene GAPDH);
(3) and (3) 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 genes according to the ROC curve by integrating the delta Ct values of the 26 genes, and determining the methylation state interpretation modes of the methylation regions of the 26 target genes.
7. Analysis of detection results
The reaction system of the kit is used for detecting 120 samples in total, wherein the samples comprise 40 endometrial cancer samples, 40 uterine fibroid samples and 40 uterine sarcoma samples.
ADCY4, ADRA1A, AMPD3, CDO1, CELF4, CHAD, CYP26C1, DRD4, FZD7, GNA12, GRM6, GYPC, HAND2, HSPA1L, HS3ST2, ITGA5, LTB4R2, MAGI2, MTMR7, ndifs 2, NEFM, PTGDR, RASSF1, SPDYA, TJP2, TRH gene methylation combined detection, statistical by logistic regression algorithm:
when the P value is 0.46-0.73, the hysteromyoma and the hysterosarcoma can be distinguished, and when the P is less than 0.46, the sample is judged to be benign; p is more than or equal to 0.46 and less than 0.73, the sample can be judged to be a malignant sample, and the very high risk is uterine sarcoma;
when P is more than or equal to 0.73, the sample can be judged to be a malignant sample, and the high risk is endometrial cancer.
Based on the above cutoff value interpretation, the detection results for the above 120 samples are: the detection specificity of the hysteromyoma is 80%, the sensitivity of the uterine sarcoma is 82.5%, and the sensitivity of the endometrial cancer is 95%.
The kit provided by the invention is verified by a small amount of samples to research a large amount of samples, and the DNA methylation is proved to have high accuracy for detecting benign and malignant lesions of endometrium, 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, performs multi-gene joint detection and function complementation, and obviously improves the detection of benign and malignant lesions of the endometrium.
The above description of the embodiments is only intended to facilitate the understanding of the core ideas of the present 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> endometrium benign and malignant lesion combined marker, detection primer probe set and kit
<130> P20220017 prereview
<160> 107
<170> SIPOSequenceListing 1.0
<210> 1
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gggagggggt cgcg 14
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gaaatcacgc tcgccgctac g 21
<210> 3
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gcgtttgtcg ttcggg 16
<210> 4
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
taggtagttt tcgggatcg 19
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gacgcactca cctaaaacgc cg 22
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gacgcactca cctaaaacgc cg 22
<210> 7
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gttgggaaaa gttgtgcg 18
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ccgccgaact tccacaccg 19
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gacgtcgggt aagttcgggc 20
<210> 10
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ataacgttta tatttttaag ttatcg 26
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gattagaccc tctactaatc cg 22
<210> 12
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
catctatttc gggcgcggag atgcgg 26
<210> 13
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
attccatgta tataaagatg gttacg 26
<210> 14
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gattaagaac tataacttaa tccg 24
<210> 15
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
atctataacg ggttcggtag tagtt 25
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
tcgagtttga agtgggagat cg 22
<210> 17
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
actacctacg ctttaaaaaa acg 23
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
acgtcggggc ggtttttacg 20
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gttaggagta ttgcggggcg 20
<210> 20
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
acttccccgc tcttcgcaaa cgc 23
<210> 21
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ttgcgcgttg ggttcgttt 19
<210> 22
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
gagggaatgg aggagggagc g 21
<210> 23
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
tacacaatta atcctcgcgt aaccg 25
<210> 24
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gtcgatcgtt tagttgttcg ttt 23
<210> 25
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
atttttagaa ttagtttttt taacg 25
<210> 26
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
aaaacccgaa ctcaaaccac c 21
<210> 27
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gtcgtgcgcg cgggcgggg 19
<210> 28
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ttgaaagaaa gaattttagt ttgggcg 27
<210> 29
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
tcgccaaaaa aaacgaacat ccc 23
<210> 30
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
tttcggcgtc gatttcgttt cga 23
<210> 31
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
agtggtcggg cgggatttcg ggcg 24
<210> 32
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
aattaactca accgacctac ccg 23
<210> 33
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
gttcggcgtt tgtaagagcg agacg 25
<210> 34
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gtgatttagg tgtcgttttt tttcg 25
<210> 35
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
aactaaccaa accgaaccaa accg 24
<210> 36
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
aggagttcgg gagcgcgatt t 21
<210> 37
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
ctattaatgg atttagagta ttaatagcg 29
<210> 38
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
tttggttata taactaataa ccaaacg 27
<210> 39
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
caatttcgtc gaattgcgcg 20
<210> 40
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
tagtgtttta tgttcgattg tatt 24
<210> 41
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
cgctaaaccc gcaaaacacc g 21
<210> 42
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
tcgggtcgtc gaatttgcgg 20
<210> 43
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
caaacaccca ttagggtagg gtgtttgcg 29
<210> 44
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
tggtggggct aaacaccccc accacg 26
<210> 45
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
aaattcggcg cgatttcgat ttgga 25
<210> 46
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
ggcgttatgg ggagtcggac 20
<210> 47
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
acccccgacc ctaaataacg 20
<210> 48
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
ttcggcgtcg attttcgtt 19
<210> 49
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
ggcgggttgg cggtttgtac 20
<210> 50
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
aaaaaccaca aaaaacgacg t 21
<210> 51
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
ggcgttggtc gacggcgcgg t 21
<210> 52
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
tttcgtttta attttttttt acg 23
<210> 53
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
tccgataaaa caaaaataac ga 22
<210> 54
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
atttcgcgtt aggacgttcg ta 22
<210> 55
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
agagagagcg agtagttttc g 21
<210> 56
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
gaccctaaaa aacccacctc c 21
<210> 57
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
ggcggcggcg taggtg 16
<210> 58
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
tttgttttag ttttagtagt agcg 24
<210> 59
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
atcgtatttc caaaaaaact caacg 25
<210> 60
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
ggcgttcgag ttaggtagga cgt 23
<210> 61
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
ggtcgtcgtt tttacggggc 20
<210> 62
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
ctatcacaac gttctaccga cc 22
<210> 63
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
tagcgtcgcg gttataaat 19
<210> 64
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
ttggttattt tttttttgtc gtttc 25
<210> 65
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
ctaacgctaa cattaaaaaa cg 22
<210> 66
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
agcgcgttag ggtcgagt 18
<210> 67
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
ggagtttgag tttattgagt tgc 23
<210> 68
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
cgttaacacg ctccaaccga ata 23
<210> 69
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
ttcgttgggc gcgttggga 19
<210> 70
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
tttgagtaat tagggtaatt acg 23
<210> 71
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
gattaaacaa ccaaaccccc gaaa 24
<210> 72
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
ttcgcgtgtg tcgggcgggg a 21
<210> 73
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
ctatcaccta cttccttaaa accg 24
<210> 74
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
ctatcaccta cttccttaaa accg 24
<210> 75
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
tcggggttcg cgcgtttaga 20
<210> 76
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
atttttagag gtggtgtaga aac 23
<210> 77
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
gaaaacgata aaaaccgaaa ccg 23
<210> 78
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
ttcgcgtcgg cgttttattt tg 22
<210> 79
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
aggttaaata tagttgttga 20
<210> 80
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
caacccaaac ccccaac 17
<210> 81
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
tagttggggg tttgggtt 18
<210> 82
<211> 79
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 82
gggagggggc cgcggcgccg ggagggaagc gcctgtcgcc cgggactctg agccagagcg 60
cagcggcgag cgtgactcc 79
<210> 83
<211> 85
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 83
caggcagctc ccgggaccga agccgggtcc acatcccccg cgcgcgagct ggtggctcag 60
cagcggcgct tcaggtgagt gcgcc 85
<210> 84
<211> 78
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 84
gctgggaaaa gttgtgcggg gaggggagga cgtcgggcaa gcccgggcgg cacggggagc 60
ggtgtggaag cccggcgg 78
<210> 85
<211> 113
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 85
gcccacactc ccaagccatc gttaaccccg ggcgcggaga tgcggagaga ccgcactcag 60
gttcgggccc tcctgacacc ttaaggaacc gtctggccgg actagcagag ggt 113
<210> 86
<211> 116
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 86
gtatataaag atggccacgt tagcaaacgg acaggctgac aacgcaagcc tcagtaccaa 60
cgggctcggc agcagcccgg gcagtgccgg gcacatgaac ggattaagcc acagcc 116
<210> 87
<211> 85
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 87
tcgagtctga agtgggagac cgacgccggg gcggctctca cgctcggcag gcgccgcaca 60
agcgcccctt taaagcgcag gcagc 85
<210> 88
<211> 87
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 88
gccaggagca ctgcggggcg caagcccggg tcagttctgc gcgttgggtt cgcccacttt 60
ctgagcgcct gcgaagagcg gggaagc 87
<210> 89
<211> 82
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 89
gagggaatgg aggagggagc ggggtcgacc gctcagctgt ccgcccagtt tcggaggcgg 60
ccacgcgagg atcaactgtg ca 82
<210> 90
<211> 118
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 90
acttctagaa ccagtctccc caacgccgtc ttcgtttcct taaaacctcg ctagagtcac 60
cccagttccc cagactccag gccgtgcgcg cgggcggggt ggcctgagcc cgggttcc 118
<210> 91
<211> 119
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 91
ctgaaagaaa gaacctcagc ctgggcgtcc aatgagaagc gccttcacct caaagatccc 60
gcctctttcg gcgtcgaccc cgccccgagg ccggaaggga tgcccgcctc ccttggcga 119
<210> 92
<211> 83
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 92
agtggtcggg cgggaccccg ggcgacaggg ttcggcgctt gtaagagcga gacggaggcc 60
cgggcaggcc ggctgagcta act 83
<210> 93
<211> 92
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 93
gtgacccagg tgccgcttcc tctcgccgcc gagggtcagg agcccgggag cgcgaccctc 60
ccccggcccg gcctggcccg gcctggccag tc 92
<210> 94
<211> 112
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 94
ggactcagag catcaacagc gccttcgccg aactgcgcga gtgcatcccc aacgtacccg 60
ccgacaccaa actctccaaa atcaagaccc tgcgcctggc caccagctac at 112
<210> 95
<211> 91
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 95
cagtgcttca tgtccgactg caccaccggg tcgccgaact tgcggccaat cagccgcttc 60
gcgtcaaaca cggtgttctg cgggttcagc g 91
<210> 96
<211> 116
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 96
atcagggcag ggtgcctgcg tctgcgtctg ggtctgtctg gtctgcatgt cggcgcgatc 60
tcgacctgga ttcgtgtccc tggatgtcga gaggccagcg tggtgggggt gtccag 116
<210> 97
<211> 129
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 97
ggcgctatgg ggagccggac gccagagtcc cctctccacg ccgtgcagct gcgctggggc 60
ccccggcgcc gacccccgct gctgccgctg ctgttgctgc tgctgccgcc gccacccagg 120
gtcgggggc 129
<210> 98
<211> 109
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 98
ggcgggctgg cggcctgcac gggggcgacc gctggcggcc acgcttgtgc tgcacctggc 60
gctggccgac ggcgcggtgc tgctgctcac gccgctcttt gtggccttc 109
<210> 99
<211> 113
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 99
ccccgcccca actccttctc acggcaggat cccgcgccag gacgctcgca gagcccgaga 60
tgtggcacca ggcgcgccac aagtccaggt cccgctattt ctgctccatc gga 113
<210> 100
<211> 99
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 100
agagagagcg agcagccctc gccccggcaa ccccaggcag aggcgcgtcg cggcggcggc 60
gcaggtgggc gcaggcgcgg aggtgggctc tctagggcc 99
<210> 101
<211> 115
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 101
cctgctccag ctctagtagc agcgtctccc caaaggcctg caagcggcac aacagcctgg 60
caggggcgcc cgagccaggc aggacgctgc cgttgagctt ctctggaaac acgat 115
<210> 102
<211> 91
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 102
ggccgccgcc tccacggggc ggggccctgg cccgggacca gcgccgcggc tataaatggg 60
ctgcggcgag gccggcagaa cgctgtgaca g 91
<210> 103
<211> 119
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 103
ctggctattt tcctcctgcc gttccgactc ggcaccagag tctgtctcta ctgagaacgc 60
agcgcgtcag ggccgagctc ttcactggcc tgctccgcgc tcttcaatgc cagcgccag 119
<210> 104
<211> 83
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 104
ggagcctgag ctcattgagc tgcgggagct ggcacccgct gggcgcgctg ggaagggccg 60
cacccggctg gagcgtgcca acg 83
<210> 105
<211> 92
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 105
tttgagtaac cagggtaacc acgctgtgcc cgcgtgtgcc gggcggggag gggaggccgc 60
agccccagcc ccgggggcct ggttgtctaa tc 92
<210> 106
<211> 71
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 106
gcgagcagct gcgtctccct cccggggctc gcgcgcccag aaccctccgg tctcaaggaa 60
gcaggtgaca g 71
<210> 107
<211> 91
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 107
atctccagag gtggtgcaga aacgaccccg cgccggcgcc ccatcctgcg gccagtgcct 60
ccgcgccccg gctccggtcc ccaccgtccc c 91

Claims (6)

1. The combined markers for benign and malignant endometrial lesions are characterized by comprising the following methylated regions of 26 target genes:
methylation region of ADCY4 gene: 82 of the amino acid sequence shown in SEQ ID NO:82,
methylation region of ADRA1A gene: the amino acid sequence shown in SEQ ID NO:83,
AMPD3 gene methylation region: 84 of the amino acid sequence shown in SEQ ID NO,
methylation region of CDO1 gene: the amino acid sequence shown in SEQ ID NO. 85,
methylation region of CELF4 gene: 86 of the amino acid sequence shown in SEQ ID NO,
methylation region of CHAD gene: the amino acid sequence shown in SEQ ID NO:87,
methylated region of CYP26C1 gene: 88 of the amino acid sequence shown in SEQ ID NO,
methylation region of DRD4 gene: 89 of the amino acid sequence shown in SEQ ID NO,
methylation region of FZD7 gene: the amino acid sequence shown in SEQ ID NO. 90,
methylation region of GNA12 gene: 91 of the amino acid sequence shown in SEQ ID NO,
methylation region of GRM6 gene: the amino acid sequence shown in SEQ ID NO. 92,
methylation region of GYPC gene: 93 of the amino acid sequence shown in SEQ ID NO,
methylation region of HAND2 gene: 94 of the amino acid sequence shown in SEQ ID NO,
methylation region of HSPA1L gene: the amino acid sequence shown in SEQ ID NO. 95,
methylation region of HS3ST2 gene: 96 of the amino acid sequence shown in SEQ ID NO:96,
methylation region of ITGA5 gene: the amino acid sequence shown in SEQ ID NO:97,
methylated region of LTB4R2 gene: the amino acid sequence shown in SEQ ID NO. 98,
methylated region of the MAGI2 gene: 99 of the amino acid sequence shown in SEQ ID NO:99,
methylation region of MTMR7 gene: the amino acid sequence shown in SEQ ID NO of 100,
methylated region of NDUFS2 gene: the amino acid sequence shown in SEQ ID NO. 101,
NEFM gene methylation region: 102 of the amino acid sequence shown in SEQ ID NO:102,
methylation region of PTGDR gene: 103 of the amino acid sequence shown in SEQ ID NO,
methylated region of RASSF1 gene: 104 of the amino acid sequence shown in SEQ ID NO:104,
methylation region of SPDYA gene: 105 of the amino acid sequence shown in SEQ ID NO,
methylation region of TJP2 gene: 106 of the amino acid sequence shown in SEQ ID NO,
methylation region of TRH gene: 107 in SEQ ID NO.
2. The primer probe set for joint detection of benign and malignant endometrial lesions, which is characterized by comprising the nucleotide sequences for correspondingly detecting the methylation states of the methylation regions of the 26 target genes according to claim 1, wherein the nucleotide sequences are as follows:
the detection primer and the probe corresponding to the methylation region of the ADCY4 gene are SEQ ID NO 1-3,
the detection primer and the probe corresponding to the ADRA1A gene methylation region are SEQ ID NO: 4-6,
the detection primers and probes corresponding to the AMPD3 gene methylation region are SEQ ID NO: 7-9,
the detection primer and the probe corresponding to the methylation region of the CDO1 gene are SEQ ID NO 10-12,
the detection primers and probes corresponding to the methylation region of the CELF4 gene are SEQ ID NO: 13-15,
the detection primer and the probe corresponding to the methylation region of the CHAD gene are SEQ ID NO: 16-18,
the detection primer and the probe corresponding to the methylation region of the CYP26C1 gene are SEQ ID NO: 19-21,
the detection primers and probes corresponding to the methylation region of the DRD4 gene are SEQ ID NO: 22-24,
the detection primer and the probe corresponding to the methylation region of the FZD7 gene are SEQ ID NO: 25-27,
the detection primer and the probe corresponding to the GNA12 gene methylation region are SEQ ID NO: 28-30,
the corresponding detection primers and probes of the methylation region of the GRM6 gene are SEQ ID NO: 31-33,
the detection primer and the probe corresponding to the methylation region of the GYPC gene are SEQ ID NO: 34-36,
the detection primers and probes corresponding to the methylation region of the HAND2 gene are SEQ ID NO: 37-39,
the detection primer and the probe corresponding to the methylation region of the HSPA1L gene are SEQ ID NO: 40-42,
the detection primer and the probe corresponding to the methylation region of the HS3ST2 gene are SEQ ID NO: 43-45,
the detection primers and probes corresponding to the methylation region of the ITGA5 gene are SEQ ID NO. 46-48,
the detection primer and the probe corresponding to the methylation region of the LTB4R2 gene are SEQ ID NO: 49-51,
the detection primers and probes corresponding to the methylated region of the MAGI2 gene are SEQ ID NO: 52-54,
the detection primer and the probe corresponding to the methylation region of the MTMR7 gene are SEQ ID NO: 55-57,
the detection primer and the probe corresponding to the methylated region of the NDUFS2 gene are SEQ ID NO: 58-60,
the detection primer and the probe corresponding to the methylation region of the NEFM gene are SEQ ID NO of 61-63,
the detection primer and the probe corresponding to the methylation region of the PTGDR gene are SEQ ID NO 64-66,
the detection primers and probes corresponding to the methylation region of the RASSF1 gene are SEQ ID NO 67-69,
the detection primers and probes corresponding to the SPDYA gene methylation region are SEQ ID NO 70-72,
the detection primer and the probe corresponding to the methylation region of the TJP2 gene are SEQ ID NO: 73-75,
the corresponding detection primers and probes of the TRH gene methylation region are SEQ ID NO: 76-78;
the 5 'end of the nucleotide sequence of the probe is marked with a fluorescent group, and the 3' end of the nucleotide sequence of the probe is marked with a quenching group.
3. The primer probe set of claim 2, further comprising a detection primer and a probe for the reference gene GAPDH, wherein the nucleotide sequence is shown in SEQ ID NO 79-81, the 5 'end of the nucleotide sequence of the detection probe for the reference gene is labeled with a fluorophore, the 3' end of the nucleotide sequence of the detection probe for the reference gene is labeled with a quencher, and the fluorophore labeled by the detection probe for the reference gene is different from the fluorophore labeled by the detection probe for the marker gene in the same detection system.
4. A kit for combined detection of benign and malignant endometrial lesions, comprising the primer probe set according to claim 2 or 3.
5. The kit according to claim 4, wherein said kit further comprises,the kit is characterized by further comprising PCR reaction liquid, wherein each PCR reaction liquid comprises 0.5-1 mu L of Taq DNA polymerase with methylation characteristic of concentration of 1U/mu L, 2-5 mu L of dNTPs with concentration of 10 mM and Mg with concentration of 2-5 mM2+2-6 μ L, 10 XDNA polymerase buffer 5 μ L and purified water to make up 15 μ L.
6. The kit according to claim 4, further comprising a sample methylation pretreatment reagent, wherein the sample methylation pretreatment reagent is a methylation detection sample pretreatment kit, docket number 20200110.
CN202210401707.8A 2022-04-18 2022-04-18 Endometrium benign and malignant lesion combined marker, detection primer probe set and kit Pending CN114480661A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210401707.8A CN114480661A (en) 2022-04-18 2022-04-18 Endometrium benign and malignant lesion combined marker, detection primer probe set and kit

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210401707.8A CN114480661A (en) 2022-04-18 2022-04-18 Endometrium benign and malignant lesion combined marker, detection primer probe set and kit

Publications (1)

Publication Number Publication Date
CN114480661A true CN114480661A (en) 2022-05-13

Family

ID=81489586

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210401707.8A Pending CN114480661A (en) 2022-04-18 2022-04-18 Endometrium benign and malignant lesion combined marker, detection primer probe set and kit

Country Status (1)

Country Link
CN (1) CN114480661A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116790761A (en) * 2023-08-22 2023-09-22 湖南宏雅基因技术有限公司 Biomarker for benign and malignant lesions of endometrium and application of biomarker
WO2024002166A1 (en) * 2022-06-29 2024-01-04 广州市基准医疗有限责任公司 Multi-gene combined fluorescence channel detection method
CN117947169A (en) * 2024-02-02 2024-04-30 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） Methylation gene marker combination for early detection of endometrial cancer, multiplex PCR detection kit and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007116417A1 (en) * 2006-04-07 2007-10-18 Silvia Sabbioni Novel dna methylation markers useful for the diagnosis of neoplastic diseases
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
CN112725438A (en) * 2021-02-05 2021-04-30 深圳市宝安区妇幼保健院 Endometrial polyp methylation marker combination, detection kit and application

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007116417A1 (en) * 2006-04-07 2007-10-18 Silvia Sabbioni Novel dna methylation markers useful for the diagnosis of neoplastic diseases
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
CN112725438A (en) * 2021-02-05 2021-04-30 深圳市宝安区妇幼保健院 Endometrial polyp methylation marker combination, detection kit and application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MICHAEL P. TRIMARCHI1等: "Identification of endometrial cancer methylation features using combined methylation analysis methods", 《PLOS ONE》 *
SHI-WEN JIANG等: "Application of DNA methylation biomarkers for endometrial cancer management", 《EXPERT REV MOL DIAGN》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024002166A1 (en) * 2022-06-29 2024-01-04 广州市基准医疗有限责任公司 Multi-gene combined fluorescence channel detection method
CN116790761A (en) * 2023-08-22 2023-09-22 湖南宏雅基因技术有限公司 Biomarker for benign and malignant lesions of endometrium and application of biomarker
CN116790761B (en) * 2023-08-22 2023-11-17 湖南宏雅基因技术有限公司 Biomarker for benign and malignant lesions of endometrium and application of biomarker
CN117947169A (en) * 2024-02-02 2024-04-30 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） Methylation gene marker combination for early detection of endometrial cancer, multiplex PCR detection kit and application thereof
CN117947169B (en) * 2024-02-02 2024-08-20 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） Methylation gene marker combination for early detection of endometrial cancer, multiplex PCR detection kit and application thereof

Similar Documents

Publication Publication Date Title
EP1456413B1 (en) Method of detection of prostate cancer
CN114480661A (en) Endometrium benign and malignant lesion combined marker, detection primer probe set and kit
CN113755603B (en) Marker, primer probe and kit for early screening and diagnosis of endometrial cancer
US20080254455A1 (en) Detecting prostate cancer
CN114277135B (en) Gastric cancer lymph node metastasis related methylation biomarker and combination and detection kit thereof
CN110484621B (en) Early warning method for liver cancer
CN113265456B (en) Primer and probe combination for detecting cervical high-grade lesion and methylation of cervical cancer related genes
CN110951871B (en) PCA3 and PSA RNA detection kit and amplification system
CN111635938B (en) Tumor detection reagent and kit
EP3625370A1 (en) Composite epigenetic biomarkers for accurate screening, diagnosis and prognosis of colorectal cancer
CN113166813A (en) Method for determining prognosis of uterine corpus carcinoma
US11840738B2 (en) Method for determining risk of urothelial carcinoma
US20230076141A1 (en) Markers, primers, probes and kit for early screening and diagnosis of endometrial cancer
CN114480655B (en) DNA methylation marker combination and application thereof, and early detection primer probe and kit for ovarian cancer
US11542559B2 (en) Methylation-based biomarkers in breast cancer screening, diagnosis, or prognosis
CN111154876B (en) Primer probe composition, kit and detection method for detecting methylation of human ADD3 and CDH23 genes
US11866790B2 (en) Combination of DNA methylation markers and use thereof, primers, probes and kit for early detection of ovarian cancer
CN117887849A (en) Methylation marker of benign and malignant endometrial lesions and application thereof
CN118685528A (en) Biomarker for methylation detection of cervical cancer genes, primer probe combination and application thereof
CN115961048A (en) Gene methylation detection primer combination, reagent and application thereof
CN118421800A (en) Product and kit for methylation detection of cervical adenocarcinoma and application of product and kit
CN118581218A (en) Nucleic acid product and kit for HPV negative cervical cancer detection and application
CN118685521A (en) Primer probe composition for detecting prostatic cancer and application thereof
CN117778582A (en) Nucleic acid combination for detecting gastric cancer, kit and application
CN111411156A (en) Kit and detection method for early detection of cancer

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20220513

RJ01 Rejection of invention patent application after publication