CN111961723A - Tumor marker for noninvasive detection of early ovarian cancer diagnosis and kit - Google Patents
Tumor marker for noninvasive detection of early ovarian cancer diagnosis and kit Download PDFInfo
- Publication number
- CN111961723A CN111961723A CN202010699975.3A CN202010699975A CN111961723A CN 111961723 A CN111961723 A CN 111961723A CN 202010699975 A CN202010699975 A CN 202010699975A CN 111961723 A CN111961723 A CN 111961723A
- Authority
- CN
- China
- Prior art keywords
- ovarian cancer
- early
- detection
- seq
- gene
- 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
Links
- 206010033128 Ovarian cancer Diseases 0.000 title claims abstract description 52
- 206010061535 Ovarian neoplasm Diseases 0.000 title claims abstract description 52
- 238000001514 detection method Methods 0.000 title claims abstract description 40
- 238000003745 diagnosis Methods 0.000 title claims abstract description 13
- 239000000439 tumor marker Substances 0.000 title claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 37
- 102100036021 WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 2 Human genes 0.000 claims abstract description 12
- 102100038080 B-cell receptor CD22 Human genes 0.000 claims abstract description 11
- 102100040126 Prokineticin-1 Human genes 0.000 claims abstract description 10
- 101000803329 Homo sapiens WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 2 Proteins 0.000 claims abstract description 9
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 claims abstract description 8
- 108010072151 Agouti Signaling Protein Proteins 0.000 claims abstract description 7
- 101000610537 Homo sapiens Prokineticin-1 Proteins 0.000 claims abstract description 7
- 210000005259 peripheral blood Anatomy 0.000 claims abstract description 6
- 239000011886 peripheral blood Substances 0.000 claims abstract description 6
- 210000001672 ovary Anatomy 0.000 claims abstract description 5
- 102000006822 Agouti Signaling Protein Human genes 0.000 claims abstract 2
- 239000000523 sample Substances 0.000 claims description 41
- 230000011987 methylation Effects 0.000 claims description 32
- 238000007069 methylation reaction Methods 0.000 claims description 32
- 101150077831 Wfikkn2 gene Proteins 0.000 claims description 7
- 101150082201 ASIP gene Proteins 0.000 claims description 6
- 101150087353 CD22 gene Proteins 0.000 claims description 5
- 101150101842 PROK1 gene Proteins 0.000 claims description 5
- 230000002611 ovarian Effects 0.000 claims description 4
- 206010028980 Neoplasm Diseases 0.000 abstract description 24
- 238000013399 early diagnosis Methods 0.000 abstract description 11
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 208000005623 Carcinogenesis Diseases 0.000 abstract description 3
- 230000036952 cancer formation Effects 0.000 abstract description 3
- 231100000504 carcinogenesis Toxicity 0.000 abstract description 3
- 108020004414 DNA Proteins 0.000 description 27
- 238000006243 chemical reaction Methods 0.000 description 13
- 238000000034 method Methods 0.000 description 8
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000007400 DNA extraction Methods 0.000 description 4
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 102100038154 Agouti-signaling protein Human genes 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 210000000918 epididymis Anatomy 0.000 description 3
- 201000010063 epididymitis Diseases 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000004393 prognosis Methods 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 230000007067 DNA methylation Effects 0.000 description 2
- GQPLMRYTRLFLPF-UHFFFAOYSA-N Nitrous Oxide Chemical compound [O-][N+]#N GQPLMRYTRLFLPF-UHFFFAOYSA-N 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000006607 hypermethylation Effects 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000011528 liquid biopsy Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000010837 poor prognosis Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241001522303 Acanthis Species 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 108010008629 CA-125 Antigen Proteins 0.000 description 1
- 102000007269 CA-125 Antigen Human genes 0.000 description 1
- 108091029523 CpG island Proteins 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 101001021410 Homo sapiens G-protein coupled receptor-associated sorting protein 1 Proteins 0.000 description 1
- 208000007571 Ovarian Epithelial Carcinoma Diseases 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 101100298837 Parengyodontium album PROK gene Proteins 0.000 description 1
- 108010010677 Phosphodiesterase I Proteins 0.000 description 1
- 102000052575 Proto-Oncogene Human genes 0.000 description 1
- 108700020978 Proto-Oncogene Proteins 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000006326 desulfonation Effects 0.000 description 1
- 238000005869 desulfonation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-N diphosphoric acid Chemical compound OP(O)(=O)OP(O)(O)=O XPPKVPWEQAFLFU-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000001808 exosome Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000004996 female reproductive system Anatomy 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000008722 morphological abnormality Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000013188 needle biopsy Methods 0.000 description 1
- 235000013842 nitrous oxide Nutrition 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000013615 primer Substances 0.000 description 1
- 239000002987 primer (paints) Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 229940005657 pyrophosphoric acid Drugs 0.000 description 1
- 238000012175 pyrosequencing Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a tumor marker and a kit for noninvasive detection of early ovarian cancer diagnosis, which have high sensitivity and strong specificity and have important clinical significance for improving the early diagnosis rate of ovarian cancer. Tumor markers for non-invasive detection of early ovarian cancer diagnosis, comprising one or more of WFIKKN2, PROK1, CD22, ASIP four genes, at least one of which is more methylated in the peripheral blood when early stage carcinogenesis occurs in human ovaries.
Description
Technical Field
The invention relates to the field of in-vitro diagnosis and detection reagents, in particular to a primer pair, a probe and a kit for multi-gene methylation joint detection for detecting early ovarian cancer and application thereof.
Background
Ovarian cancer is one of the most common malignancies in the female reproductive system. Ovarian cancer is derived from a variety of tissues, such as epithelial, erotic-mesenchymal, germ cells, and the like. Epithelial ovarian cancer is most common and accounts for 90% of all. Ovarian cancer is currently discovered primarily by imaging and detection of blood tumor markers. However, ovarian cancer, which is detected by these two tests, generally has progressed to a middle or advanced stage and has a poor prognosis. The poor prognosis of ovarian cancer is strongly related to the late discovery, and the ovarian cancer is frequently found in the late stage because the ovarian cancer is developed in the abdominal cavity, has latent onset and insignificant symptoms and is difficult to attach importance. Despite the existing various treatment methods such as surgery, radiotherapy and chemotherapy, the 5-year survival rate of ovarian cancer is still not high. At the end, the early diagnosis technology is still lack, the tumors cannot be found early, and the improvement of the ovarian cancer screening level is urgently needed in the clinical aspect. Early diagnosis of ovarian cancer, early treatment, is an important means to improve prognosis.
The tumor marker carbohydrate antigen CA125 has high sensitivity but not high enough specificity, and CA125 can be increased in ovarian cancer and ovarian benign diseases. Therefore, it sometimes causes false positive of the detection result. Human epididymis protein HE4 is first found in human epididymis epithelial cells and then used as a novel tumor marker of ovarian cancer, and the specificity of the human epididymis protein HE4 in distinguishing ovarian tumors is higher than that of CA 125. Tumor markers of ovarian cancer have limitations of sensitivity and specificity, and no index can have high sensitivity and specificity to ovarian cancer at the same time. In addition, no tumor markers could be detected well for early stage ovarian cancer. Ultrasonic detection is a main imaging means for finding ovarian cancer, but the detection rate of the stage I ovarian cancer without ovary enlargement or morphological abnormality by ultrasonic is not high.
The liquid biopsy is one of the hot techniques in the field of early tumor screening and detection at present, and can detect the whole body tumor information by collecting a small amount of blood samples. The method avoids the limitations of traditional operations and needle biopsy, and is a breakthrough technology for detecting tumors and cancers. Liquid biopsies mainly include CTC and ctDNA and exosome detection. ctDNA, also called circulating tumor DNA, is a DNA fragment released from tumor cells into the blood circulation. The mutation property and the type of the gene are consistent with those of tumor tissues, so that the property, the stage, the prognosis judgment, the targeted treatment and the like of the tumor can be evaluated by detecting ctDNA. The detection content of the ctDNA comprises genomics, transcriptomics, epigenomics and the like, and the detection of single gene can be carried out, and the detection of multiple genes can also be carried out. Since ctDNA is derived from tumor tissue, it contains genetic information that is consistent throughout the tumor tissue.
On a normal genome, 70% to 80% of gene cpgs are methylated, whereas the cpgs of the promoter region are unmethylated. In tumor tissue, the opposite is true, showing hypomethylation of the whole genome and hypermethylation of the promoter region. The hypomethylation of the whole genome causes the instability of the genome and can activate silent genes such as protooncogenes under normal conditions, and the hypermethylation of a promoter region can inhibit and silence the expression of cancer suppressor genes, DNA repair genes, cell cycle regulatory genes and apoptosis genes, thereby promoting the occurrence of tumors. DNA methylation is therefore closely associated with the development of a tumor, an early event in tumorigenesis. Since CPG islands are highly methylated earlier than tumor hyperplasia, the methylation sites of certain genes can be used as biomarkers for early diagnosis of tumors. Abnormal methylation of the genes can be used as an early diagnosis marker of ovarian cancer.
In the past, methylation research focuses on single-gene promoter region methylation, and recent research finds that the combination of multiple genes can further improve the specificity and sensitivity of DNA methylation characteristics. The characteristic methylation fingerprint maps in different tumor tissues can be used for early diagnosis and staging of tumors, curative effect evaluation, relapse monitoring, prognosis judgment and the like. In recent years, ovarian cancer and gene abnormal methylation have been studied more frequently, but many gene methylation combination (panel) detection studies and reports are few.
Disclosure of Invention
Based on the current situation of early diagnosis of ovarian cancer, in order to overcome the defects of the prior art, the tumor marker and the kit for noninvasive detection of early diagnosis of ovarian cancer provided by the invention have the advantages of high sensitivity and strong specificity, and have important clinical significance for improving the early diagnosis rate of ovarian cancer.
Tumor markers for non-invasive detection of early ovarian cancer diagnosis, comprising one or more of WFIKKN2, PROK1, CD22, ASIP four genes, at least one of which is more methylated in the peripheral blood when early stage carcinogenesis occurs in human ovaries.
The tumor marker has the advantage that when the human ovarian has early canceration, the methylation degree of the WFIKKN2 gene in peripheral blood is increased.
When the human ovary has early canceration, the methylation degree of at least two genes in the four genes is improved.
A noninvasive multi-gene combined detection kit for early ovarian cancer diagnosis comprises a plurality of primer pairs and probes, including primer pairs and probes for detecting WFIKKN2, PROK1, CD22 and ASIP gene methylation and primer pairs and probes of internal reference ACTB, wherein the sequences of the primers and the probes are shown as SEQ ID No: 1-SEQ ID No. 15.
The invention has the beneficial effects that:
has high sensitivity and high specificity, and is suitable for early noninvasive screening of human ovarian cancer.
Detailed Description
The invention is further illustrated by the following examples.
The invention provides a primer pair and a probe for noninvasive multi-gene methylation combined detection of early ovarian cancer, which comprise a primer pair and a probe for detecting WFIKKN2, PROK1, CD22 and ASIP gene methylation respectively. The WFIKKN2 gene is also called growth differentiation factor related serum protein 1(GASP1), belongs to FS family, PROK1 gene prodynein gene, CD22 gene belongs to cell differentiation antigen, and ASIP gene is acanthis signal protein gene.
Example 1
This example is to screen 4 candidate genes that are abnormally methylated in ovarian cancer by applying bioinformatics and experimental verification methods, and to verify the detection accuracy of the candidate genes in ovarian cancer in plasma samples.
Validation of methylation of candidate genes by Pyrophosphoric acid sequencing experiments
After 10 ovarian cancer patient plasma samples were PCR amplified, the amplification products were pyrosequenced using pyrosequencing. The results show that the average methylation rates of WFIKKN2, PROK1, CD22 and ASIP genes reach more than 85 percent, the average methylation rates of the control groups are less than 5 percent, and the differences are significant compared with the control groups. The methylation of the 4 genes is shown to have a remarkable difference between the ovarian cancer and the normal group, and can be used as an early diagnosis marker of the ovarian cancer.
WFIKKN2 was highly specific among the 10 samples.
Example 2
Based on the results of example 1, this example designed gene-specific primer pairs and probes for early detection of ovarian cancer, including primers and probes for detection of WFIKKN2, PROK1, CD22, ASIP gene methylation, and primers and probes for internal reference ACTB, respectively; wherein the primer pair for detecting the methylation of the WFIKKN2 gene is SEQ ID No:1 and SEQ ID No: 2, and the probe is SEQ ID No: 3 sequence (b).
WFIKKN2F1: 5’- GTTTGTTTAAGGGTTGTGTGAAG-3’ （SEQ ID No：1）；
WFIKKN2R1: 5’- ACCATTCTACAAAATCTATCTTTTCTACTT -3’（SEQ ID No：２）；
WFIKKN2 probe: TGATGAAATTAAGGGGAATTTAAA (SEQ ID No: 3).
The primer pair for detecting methylation of the PROK1 gene is SEQ ID No: 4 and SEQ ID No: 5, the probe is SEQ ID No: 6 sequence.
PROK1F1: 5’- GGGGATGTAGGAGAGTTTGGTT -3’（SEQ ID No：４）；
PROK1R1: 5’- CCCCTATAATCACAACACAATCAAACAC -3’（SEQ ID No：５）；
Probe for PROK 1: AGTTTGTTAGGTATAAGGT (SEQ ID No: 6).
The primer pair for detecting the methylation of the CD22 gene is SEQ ID No: 7 and SEQ ID No: 8, and the probe is SEQ ID No: 9 sequence.
CD22F1: 5’- AGTTTAGTGGGTGAGTAAGAAAAG-3’（SEQ ID No：７）；
CD22R1: 5’- AATACTAACCCAAACCACAATCC -3’（SEQ ID No：８）；
CD22 probe: GGGTGAGTAAGAAAAGT (SEQ ID No: 9).
The primer pair for detecting the methylation of the ASIP gene is SEQ ID No: 10 and SEQ ID No: 11, and the probe is SEQ ID No: 12 sequence.
ASIPF1: 5’- TAGGAGGTTGAGGTAGGAGAAT -3’（SEQ ID No：１０）；
ASIPR1: 5’- ACCCACAAAACCATAATTTATTATCA -3’（SEQ ID No：１１）；
ASIP probe: GAGGTAGGAGAATGG (SEQ ID No: 12).
The primer pair for detecting methylation of the ACTB reference gene is SEQ ID No: 13 and SEQ ID No: 14, and the probe is a sequence shown in SEQ ID No:15 sequence (b).
ACTBF1: 5’-AAGACCTGTACGMCAACAC-3’（SEQ ID No：１３）；
ACTBR1: 5’-GGAGCAATGATCTTGATCTT-3’（SEQ ID No：１４）；
ACTB probe: TCTGGCGGCACCACCATGTACC (SEQ ID No: 15).
The 5 'end of the gene specific probe is combined with fluorescent dye, and the 3' end is combined with quencher. When the probe is paired with the target sequence, the fluorescence emitted by the fluorophore is quenched by proximity to a quencher at the 3' end. When the extension reaction is performed, the 5' exonuclease activity of the polymerase cleaves the probe, so that the fluorescent group is separated from the quencher and fluorescence is emitted. The production of a molecule is accompanied by the generation of a fluorescent signal for a molecule. The released fluorophores accumulate as the number of amplification cycles increases.
The fluorescent dye may be selected from the group consisting of FAM, ROX, CY5, and HEX in combination. Such as FAM fluorescent dyes. The gene-specific probe is different from the internal reference probe in the 5' end fluorescent group.
The 5' end fluorescent group of the internal reference probe can be a VIC fluorescent group, so that the fluorescent signals of the gene to be detected and the internal reference gene can be conveniently distinguished, and the detection result can be conveniently judged.
The 3' ends of the gene specific probe and the internal reference probe are both provided with quenching groups, and the quenching groups are MGB and TAMRA.
Example 3
This example is a multigene methylation detection kit for early ovarian cancer screening comprising the primer pair and probe described in example 1.
For those skilled in the art, the kit may further comprise a free DNA extraction reagent, a sulfite conversion reagent, a real-time fluorescent quantitative PCR amplification reagent, a negative and positive control, and the like. As the free DNA extraction kit and the sulfite conversion reagent, commercially available kits can be used. The fluorescent quantitative PCR reagent comprises PCR amplification buffer solution, primers, probes, DNA polymerase and the like. The positive control can be human genomic DNA and the negative control can be deionized water.
Example 4
This example is a method for screening and detecting early ovarian cancer by using the above-mentioned kit.
1) Materials, kits and apparatus
A free DNA extraction kit, a sulfite conversion kit and a fluorescent quantitative PCR instrument.
2) Sample preparation
Peripheral plasma, negative and positive controls of the patients were thawed for use. The patient was admitted with a large informed consent.
3) Extraction of free DNA and sulfite conversion
And (3) extracting free DNA from the plasma sample according to the DNA extraction specification, wherein the process comprises DNA cracking and combination, DNA washing and elution, and finally, eluting the DNA by using an eluent to obtain the extracted DNA.
The sulfite conversion experiments were performed as described, and included sulfite conversion, binding step, first wash, desulfonation, second wash, third wash, fourth wash, drying, and elution of DNA.
4, PCR amplification reaction of the sample
Sample adding, wherein 15ul of sample template DNA, negative quality control substances and positive quality control substances are respectively added into a PCR reaction tube, 35ul of PCR reaction solution is added, and the total amount of a reaction system is 50 ul.
The reaction condition is 95 ℃ denaturation for 5 min; annealing and extending at 95 ℃ for 10s and 60 ℃ for 35s for 45 cycles;
performing fluorescent signal and result judgment on the DNA sample subjected to PCR amplification, and if the internal reference reaction is normal, judging that the suspected ovarian cancer case is a suspicious ovarian cancer case if any one of the four genes WFIKKN2, PROK1, CD22 and ASIP is positive in the PCR amplification, and performing follow-up detection or B-ultrasonic confirmation; two or more genes are positive, the result is judged to be positive, and confirmation diagnosis such as B-ultrasonic diagnosis or CT diagnosis is recommended.
Compared with the existing ovarian cancer detection technology, the invention has the following advantages and characteristics:
the abnormal gene methylation is earlier than the tumor lump formation, so the screening and detection of the tumor are also earlier than the detection of the existing tumor marker.
According to the invention, the gene methylation detection technology is adopted to accurately detect the methylation sites related to the ovarian cancer, the sensitivity is high, the specificity is good, the accuracy is high, the gene methylation detection with extremely low copy number can be realized, and the lesion signs of the ovarian cancer can be prompted at an early stage.
The detection method established according to the invention has better specificity on ovarian cancer and has no cross reaction on normal samples and blank controls.
The detection result of the clinical sample shows that hypermethylated genes are not detected in 10 normal samples, and more than 85% of genes are detected in 10 ovarian cancer samples. P <0.001 compared to the normal group. Showing the accuracy of the detection kit and method.
As can be seen from the above examples, the kit and the method of the present invention are suitable for detecting methylation detection of WFIKKN2, PROK1, CD22 and ASIP genes in peripheral blood, and the detection result provides detection basis for early diagnosis of ovarian cancer.
The embodiments of the present invention have been described in detail, but this is only for exemplary purposes, and the present invention is not limited to the above-described embodiments.
Sequence listing
<110> Panzhi text
Xulauhong (red of laughing gas)
<120> tumor marker and kit for noninvasive detection of early ovarian cancer diagnosis
<141> 2020-07-17
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gtttgtttaa gggttgtgtg aag 23
<210> 2
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
accattctac aaaatctatc ttttctactt 30
<210> 3
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tgatgaaatt aaggggaatt taaa 24
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ggggatgtag gagagtttgg tt 22
<210> 5
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cccctataat cacaacacaa tcaaacac 28
<210> 6
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
agtttgttag gtataaggt 19
<210> 7
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
agtttagtgg gtgagtaaga aaag 24
<210> 8
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
aatactaacc caaaccacaa tcc 23
<210> 9
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gggtgagtaa gaaaagt 17
<210> 10
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
taggaggttg aggtaggaga at 22
<210> 11
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
acccacaaaa ccataattta ttatca 26
<210> 12
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gaggtaggag aatgg 15
<210> 13
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
aagacctgta cgmcaacac 19
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ggagcaatga tcttgatctt 20
<210> 15
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
tctggcggca ccaccatgta cc 22
Claims (4)
1. A tumor marker for noninvasive detection of early ovarian cancer diagnosis, which is characterized in that,
comprises one or more of WFIKKN2, PROK1, CD22 and ASIP, and the methylation degree of at least one of the four genes in the peripheral blood is increased when early canceration of human ovarian is generated.
2. The tumor marker of claim 1, wherein the WFIKKN2 gene methylation level in peripheral blood is increased when early stage canceration occurs in human ovaries.
3. The tumor marker of claim 1, wherein at least two of said four genes are methylated to an increased extent when early stage canceration of a human ovarian is present.
4. A noninvasive multi-gene combined detection kit for early ovarian cancer diagnosis is characterized by comprising a plurality of primer pairs and probes, including the primer pairs and probes for detecting WFIKKN2, PROK1, CD22 and ASIP gene methylation and the primer pairs and probes of internal reference ACTB, wherein the sequences of the primers and probes are shown as SEQ ID No: 1-SEQ ID No. 15.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010699975.3A CN111961723A (en) | 2020-07-17 | 2020-07-17 | Tumor marker for noninvasive detection of early ovarian cancer diagnosis and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010699975.3A CN111961723A (en) | 2020-07-17 | 2020-07-17 | Tumor marker for noninvasive detection of early ovarian cancer diagnosis and kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111961723A true CN111961723A (en) | 2020-11-20 |
Family
ID=73361962
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010699975.3A Pending CN111961723A (en) | 2020-07-17 | 2020-07-17 | Tumor marker for noninvasive detection of early ovarian cancer diagnosis and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111961723A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113718034A (en) * | 2021-09-27 | 2021-11-30 | 中国医学科学院肿瘤医院 | Marker, detection kit and detection method for guiding medication and curative effect evaluation of ovarian cancer platinum drug-resistant patient |
| 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 |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1553190A (en) * | 2003-06-05 | 2004-12-08 | 旭 马 | Four mucin enzyme linked immunosorbent assay reagent box for early diagnosing oophoroma |
| US20080038754A1 (en) * | 2004-07-14 | 2008-02-14 | The Regents Of The University Of California | Biomarkers for Early Detection of Ovarian Cancer |
| US20100216137A1 (en) * | 2007-04-05 | 2010-08-26 | SOURCE PRECISION MEDICINE, INC d/b/a SOURCE MDX | Gene Expression Profiling for Identification, Monitoring and Treatment of Ovarian Cancer |
| CN104422768A (en) * | 2013-08-30 | 2015-03-18 | 广州瑞博奥生物科技有限公司 | Antibody chip kit for joint detection of early ovarian cancer markers |
| KR20150050776A (en) * | 2013-10-31 | 2015-05-11 | 안국약품 주식회사 | Biomarker combination for diagnosis of ovarian cancer |
| US20160340743A1 (en) * | 2014-02-10 | 2016-11-24 | Baylor College Of Medicine | Recurrent fusion genes identified in high -grade serous ovarian carcinoma |
| WO2019238944A1 (en) * | 2018-06-15 | 2019-12-19 | Olink Proteomics Ab | Biomarker panel for ovarian cancer |
| CN110716042A (en) * | 2019-10-23 | 2020-01-21 | 郑州大学 | Serum protein marker, kit and detection method for early screening and diagnosis of ovarian cancer |
-
2020
- 2020-07-17 CN CN202010699975.3A patent/CN111961723A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1553190A (en) * | 2003-06-05 | 2004-12-08 | 旭 马 | Four mucin enzyme linked immunosorbent assay reagent box for early diagnosing oophoroma |
| US20080038754A1 (en) * | 2004-07-14 | 2008-02-14 | The Regents Of The University Of California | Biomarkers for Early Detection of Ovarian Cancer |
| US20100216137A1 (en) * | 2007-04-05 | 2010-08-26 | SOURCE PRECISION MEDICINE, INC d/b/a SOURCE MDX | Gene Expression Profiling for Identification, Monitoring and Treatment of Ovarian Cancer |
| CN104422768A (en) * | 2013-08-30 | 2015-03-18 | 广州瑞博奥生物科技有限公司 | Antibody chip kit for joint detection of early ovarian cancer markers |
| KR20150050776A (en) * | 2013-10-31 | 2015-05-11 | 안국약품 주식회사 | Biomarker combination for diagnosis of ovarian cancer |
| US20160340743A1 (en) * | 2014-02-10 | 2016-11-24 | Baylor College Of Medicine | Recurrent fusion genes identified in high -grade serous ovarian carcinoma |
| WO2019238944A1 (en) * | 2018-06-15 | 2019-12-19 | Olink Proteomics Ab | Biomarker panel for ovarian cancer |
| CN110716042A (en) * | 2019-10-23 | 2020-01-21 | 郑州大学 | Serum protein marker, kit and detection method for early screening and diagnosis of ovarian cancer |
Non-Patent Citations (1)
| Title |
|---|
| 许沈华: "肿瘤标志物在恶性肿瘤诊断与治疗的应用" * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113718034A (en) * | 2021-09-27 | 2021-11-30 | 中国医学科学院肿瘤医院 | Marker, detection kit and detection method for guiding medication and curative effect evaluation of ovarian cancer platinum drug-resistant patient |
| 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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113215264B (en) | Detection kit for early screening of TMEM101 gene methylation in human peripheral blood circulating tumor DNA (deoxyribonucleic acid) of endometrial cancer | |
| CN109504780B (en) | DNA methylation qPCR kit for lung cancer detection and use method thereof | |
| TWI730429B (en) | HOXA7 methylation detection reagent | |
| CN111961723A (en) | Tumor marker for noninvasive detection of early ovarian cancer diagnosis and kit | |
| EP2304057A2 (en) | Method for the detection of ovarian cancer | |
| TWI789551B (en) | Application of HOXA9 methylation detection reagent in the preparation of lung cancer diagnostic reagent | |
| WO2020063901A1 (en) | Use of hoxa7 and hoxa9 methylation detection reagent in preparing lung cancer diagnostic reagent | |
| CN113621704B (en) | Reagent and kit for detecting and diagnosing liver cancer | |
| US11130998B2 (en) | Unbiased DNA methylation markers define an extensive field defect in histologically normal prostate tissues associated with prostate cancer: new biomarkers for men with prostate cancer | |
| CN113637745B (en) | Methylated molecular markers for detecting benign and malignant lung nodules or combination and application thereof | |
| WO2020063898A1 (en) | Use of hoxa7 methylation detection reagent in preparation of diagnostic reagent for lung cancer | |
| JPWO2020116573A1 (en) | How to determine the prognosis of endometrial cancer | |
| CN114891886B (en) | Nucleic acid product, kit and application for diagnosing bladder cancer | |
| TWI789550B (en) | HOXA9 Methylation Detection Reagent | |
| CN114150065B (en) | Marker for colorectal cancer or precancerous lesion and application thereof | |
| CN117467763A (en) | Nucleic acid combination and kit for detecting methylation level of non-small cell lung cancer biomarker | |
| CN117106899A (en) | Primer pair, primer probe combination, kit and application for detecting benign and malignant lung nodule | |
| CN117187388A (en) | Application of GRIK2 gene as marker in preparation of lung cancer detection kit | |
| CN117248021A (en) | Nucleic acid combination, kit and application for detecting urothelial cancer or precancerous lesions | |
| CN116479129A (en) | Nucleic acid combination for liver cancer detection, kit and application | |
| CN117265110A (en) | Kit for detecting lung squamous carcinoma and application thereof | |
| CN117778572A (en) | Nucleic acid combination for detecting thyroid cancer, detection kit and application | |
| CN117778573A (en) | Nucleic acid combination for detecting thyroid cancer biomarker, kit and application | |
| CN117327794A (en) | Reagent and kit for detecting benign and malignant lung nodules and application of reagent and kit | |
| CN117587125A (en) | Application of reagent for detecting methylation in preparation of pancreatic cancer diagnosis product |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20201120 |
|
| WD01 | Invention patent application deemed withdrawn after publication |