CN114250302B - Composition and kit for detecting cervical intraepithelial neoplasia and application - Google Patents
Composition and kit for detecting cervical intraepithelial neoplasia and application Download PDFInfo
- Publication number
- CN114250302B CN114250302B CN202111594414.8A CN202111594414A CN114250302B CN 114250302 B CN114250302 B CN 114250302B CN 202111594414 A CN202111594414 A CN 202111594414A CN 114250302 B CN114250302 B CN 114250302B
- Authority
- CN
- China
- Prior art keywords
- gene
- cervical
- hpv
- intraepithelial neoplasia
- target sequence
- 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.)
- Active
Links
- 206010008263 Cervical dysplasia Diseases 0.000 title claims abstract description 46
- 208000007951 cervical intraepithelial neoplasia Diseases 0.000 title claims abstract description 46
- 239000000203 mixture Substances 0.000 title claims abstract description 29
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 49
- 101100109973 Homo sapiens ACTR3B gene Proteins 0.000 claims abstract description 16
- 238000003780 insertion Methods 0.000 claims abstract description 16
- 230000037431 insertion Effects 0.000 claims abstract description 16
- 239000012472 biological sample Substances 0.000 claims abstract description 15
- 208000006374 Uterine Cervicitis Diseases 0.000 claims abstract description 8
- 206010008323 cervicitis Diseases 0.000 claims abstract description 8
- 239000000523 sample Substances 0.000 claims description 39
- 108020004414 DNA Proteins 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 17
- 239000012634 fragment Substances 0.000 claims description 16
- 101150012822 DOCK3 gene Proteins 0.000 claims description 13
- 101100387908 Homo sapiens DOCK3 gene Proteins 0.000 claims description 13
- 238000001514 detection method Methods 0.000 claims description 13
- 238000009396 hybridization Methods 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- 101000866238 Homo sapiens Dedicator of cytokinesis protein 3 Proteins 0.000 claims description 8
- 239000011324 bead Substances 0.000 claims description 8
- 101000728742 Homo sapiens Actin-related protein 3B Proteins 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 5
- 102100029631 Actin-related protein 3B Human genes 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 102000053602 DNA Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108010090804 Streptavidin Proteins 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims description 2
- 230000003902 lesion Effects 0.000 abstract description 14
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 9
- 238000007672 fourth generation sequencing Methods 0.000 abstract description 7
- 238000007480 sanger sequencing Methods 0.000 abstract description 4
- 238000012795 verification Methods 0.000 abstract description 2
- 241000701806 Human papillomavirus Species 0.000 description 43
- 238000012163 sequencing technique Methods 0.000 description 22
- 210000001519 tissue Anatomy 0.000 description 14
- 230000010354 integration Effects 0.000 description 11
- 206010008342 Cervix carcinoma Diseases 0.000 description 8
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 8
- 201000010881 cervical cancer Diseases 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 102100031604 Dedicator of cytokinesis protein 3 Human genes 0.000 description 5
- 206010061218 Inflammation Diseases 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000004054 inflammatory process Effects 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 108091092584 GDNA Proteins 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 208000009608 Papillomavirus Infections Diseases 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 210000003679 cervix uteri Anatomy 0.000 description 3
- 201000003565 cervix uteri carcinoma in situ Diseases 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- -1 fatty acid ester Chemical class 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 208000020082 intraepithelial neoplasia Diseases 0.000 description 3
- 238000010801 machine learning Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 201000010153 skin papilloma Diseases 0.000 description 3
- 208000022159 squamous carcinoma in situ Diseases 0.000 description 3
- 208000022625 uterine cervix carcinoma in situ Diseases 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 2
- 101001123298 Homo sapiens PR domain zinc finger protein 14 Proteins 0.000 description 2
- 241000341655 Human papillomavirus type 16 Species 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 102100028974 PR domain zinc finger protein 14 Human genes 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 210000002469 basement membrane Anatomy 0.000 description 2
- 238000004422 calculation algorithm Methods 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 210000005081 epithelial layer Anatomy 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000003205 genotyping method Methods 0.000 description 2
- 230000006607 hypermethylation Effects 0.000 description 2
- 206010020718 hyperplasia Diseases 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000007637 random forest analysis Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 206010061424 Anal cancer Diseases 0.000 description 1
- 208000007860 Anus Neoplasms Diseases 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 208000009458 Carcinoma in Situ Diseases 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 208000032271 Malignant tumor of penis Diseases 0.000 description 1
- 208000003445 Mouth Neoplasms Diseases 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 208000002471 Penile Neoplasms Diseases 0.000 description 1
- 206010034299 Penile cancer Diseases 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- 208000019802 Sexually transmitted disease Diseases 0.000 description 1
- 206010044002 Tonsil cancer Diseases 0.000 description 1
- 208000006842 Tonsillar Neoplasms Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 206010047741 Vulval cancer Diseases 0.000 description 1
- 208000004354 Vulvar Neoplasms Diseases 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 201000011165 anus cancer Diseases 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- JJWKPURADFRFRB-UHFFFAOYSA-N carbonyl sulfide Chemical compound O=C=S JJWKPURADFRFRB-UHFFFAOYSA-N 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 238000002573 colposcopy Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000002380 cytological effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 201000004306 epidermodysplasia verruciformis Diseases 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 210000004392 genitalia Anatomy 0.000 description 1
- 201000004933 in situ carcinoma Diseases 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 208000012987 lip and oral cavity carcinoma Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 201000002120 neuroendocrine carcinoma Diseases 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 206010038038 rectal cancer Diseases 0.000 description 1
- 201000001275 rectum cancer Diseases 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 210000005000 reproductive tract Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000013024 troubleshooting Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 201000005102 vulva cancer Diseases 0.000 description 1
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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/112—Disease subtyping, staging or classification
-
- 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
- C12Q2600/158—Expression 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 composition, a kit and an application for detecting cervical intraepithelial neoplasia, wherein the composition comprises a reagent capable of indicating the ACTR3B gene state in a biological sample, and when the existence of HPV gene insertion in or near the ACTR3B gene is detected, the cervical state is diagnosed as the cervical intraepithelial neoplasia. The invention determines the cervical intraepithelial neoplasia specific gene based on nanopore sequencing, and further performs reliability verification through Sanger sequencing. Therefore, the invention provides an effective means for diagnosing or identifying cervical lesions, colposcopic shunt, whether CIN I level intervenes or not and the like, particularly distinguishing cervicitis and cervical CIN.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a composition for detecting cervical intraepithelial neoplasia, a kit and application.
Background
Cervical Intraepithelial Neoplasia (CIN) belongs to cervical epithelial dysplasia, which is a dynamically changing process classified into three levels, i.e., low, medium and high, i.e., level I, II and III. High CIN refers to atypical lesions that involve the entire epithelial layer but do not infiltrate the basement membrane, also known as carcinoma in situ of the cervix. The continuous development can infiltrate into the basement membrane, and can form cervical infiltration cancer. Low grade CIN lesions refer to cervical epithelial dysplasia that only affects the upper 1/3 of the entire epithelial layer. CIN is a dynamically changing process, but timely treatment can reverse and return to normal. Therefore, the diagnosis of CIN is of great significance for preventing or early intervening in cervical cancer.
Currently, CIN is mainly a cytometric examination. Specifically, exfoliated cells of the cervical orifice are collected through a liquid-based thin-layer cell kit, a full-automatic thin-layer cell pelleter is used for flaking, and cytological classification diagnosis is carried out according to the form of cell nuclei to judge whether cancerous lesions exist. The examination means is poor in accuracy and specificity for CIN, missing examination often occurs, and since CIN is similar to a lesion of cervicitis, the two cannot be well distinguished by cytology.
In recent years, with the research on the relationship between Human Papilloma Virus (HPV) infection and the lower genital tract, it has been found that HPV infection is associated with the occurrence of CIN. HPV infection, a specific type of sexually transmitted disease, is the etiological agent for CIN and cervical cancer development. Molecular biology and epidemiological studies have shown that human papillomaviruses are oncogenic. HPV can be divided into different types according to their oncogenicity: the HPV16, 18, 45 and 56 are high-risk types, 11 types of HPV31, 33, 35 and the like are medium-risk types, and 8 types of HPV6, 11, 26 and the like are low-risk types. Therefore, the search for specific biomarkers of CIN is of great significance.
CN104870653A discloses molecular diagnostic markers PRDM14 and FAM19a4 for HPV-induced invasive cancers and high-grade precursor lesions thereof, which comprise detecting hypermethylation in PRDM14 and/or FAM19a4 genes in cells, whereas such hypermethylation indicates the presence of HPV-induced precursor lesions and HPV-induced invasive cancers with invasive potential.
However, there are currently no molecular markers for effectively distinguishing CIN from early lesions.
The information in this background is only for the purpose of illustrating the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is known to a person skilled in the art.
Disclosure of Invention
On the basis of collecting a TCT sample without intraepithelial neoplasia due to cervical high-risk HPV infection, TCT or fresh tissue samples of cervical lesions CIN I, CIN II and CIN III and TCT or fresh tissue samples of cervical cancer, the invention utilizes the three-generation nanopore technology and detects HPV DNA integration positions based on the probe hybridization capture principle to obtain HPV integration information in different stage samples, and further analyzes to obtain a specific marker which can well distinguish cervicitis and CIN and is used for assisting clinical early diagnosis and treatment. Specifically, the present invention includes the following.
In a first aspect of the invention, there is provided a composition for cervical intraepithelial neoplasia detection comprising an agent capable of indicating the status of the ACTR3B gene in a biological sample.
According to the composition for detecting cervical intraepithelial neoplasia of the present invention, preferably, the biological sample is derived from cervical tissue of an HPV infected subject.
The composition for detecting cervical intraepithelial neoplasia according to the present invention preferably further comprises an agent capable of indicating the status of DOCK3 gene in a biological sample.
According to the composition for cervical intraepithelial neoplasia detection of the present invention, preferably, the reagent comprises a barcode linker and a barcode primer, and the status of the DOCK3 gene or ACTR3B gene is indicated by a method comprising the steps of:
a. collecting a biological sample from a subject, and separating DNA to obtain a DNA fragment with the length of 1-3 Kb;
b. separating a target sequence from the DNA fragment, constructing a library, connecting a bar code joint at the tail end, and enriching by adopting a bar code primer to enable the target sequence or part of the target sequence to pass through a nanopore located in a chip, wherein the chip is arranged near an electrode, the electrode can detect current passing through the nanopore, and information of the target sequence is detected through current change.
According to the composition for cervical intraepithelial neoplasia detection of the present invention, preferably, the biological sample is exfoliated cells, surgical or punctured tissue, and step a comprises adjusting the ultrasound frequency and energy and/or optimizing the disruption time and system to disrupt DNA in the biological sample to a length of 1-3 Kb.
The composition for detecting cervical intraepithelial neoplasia according to the invention preferably further comprises a capture probe for enriching the target sequence.
In a second aspect of the present invention, there is provided use of an agent in the preparation of a composition for cervical intraepithelial neoplasia detection by a method comprising the steps of, wherein the agent comprises an agent capable of indicating the status of the ACTR3B gene in a biological sample, the method comprising:
a. collecting a biological sample from a subject, and separating DNA to obtain a DNA fragment with the length of 1-3 Kb;
b. separating a target sequence from the DNA fragment, constructing a library, connecting a bar code joint at the tail end, and enriching by adopting a bar code primer to enable the target sequence or part of the target sequence to pass through a nanopore located in a chip, wherein the chip is arranged near an electrode, the electrode can detect current passing through the nanopore, and information of the target sequence is detected through current change.
According to the application, preferably, the enrichment comprises the steps of mixing a DNA fragment with the length of 1-3Kb with human cot-1DNA, evaporating to dryness, redissolving in an optimized hybridization solution, incubating at room temperature, keeping at 93-97 ℃ for 5-15 minutes, adding a probe set consisting of a plurality of probes, slowly cooling to the hybridization temperature by reducing the temperature by 0.1 ℃ per minute to improve the specificity of a captured target sequence and the binding stability of the long fragment, hybridizing at 63-67 ℃ for 4-16 hours, mixing a product with streptavidin magnetic beads, incubating for 45 minutes, washing the magnetic beads with a washing solution, and synthesizing and enriching by using a random primer, dNTP and an enzyme with single-stranded DNA as a guide strand synthesis activity.
According to the use of the present invention, preferably, the cervical state is diagnosed as cervical intraepithelial neoplasia when the presence of HPV gene insertion is detected inside or in the vicinity of ACTR3B gene.
According to the use of the present invention, preferably, the cervical state is diagnosed as cervicitis when the presence of HPV gene insertion is detected within or near the DOCK3 gene.
In a third aspect of the invention, there is provided a kit comprising a composition according to the first aspect and instructions for using the composition for detection.
The invention provides an effective means for diagnosing or identifying cervical lesions, colposcopic shunt, interference of CIN I grade and the like, particularly distinguishing cervicitis and cervical CIN.
Drawings
Figure 1 is a histogram of qPCR results in different tissues for the DOCK3 gene.
Figure 2 is a histogram of qPCR results in different tissues for the ACTR3B gene.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
The composition, kit and use for detecting cervical intraepithelial neoplasia of the present invention are described below.
Composition comprising a fatty acid ester and a fatty acid ester
The compositions of the present invention include agents capable of indicating the status of the DOCK3 gene in a biological sample, examples of which include, but are not limited to, barcode linkers and barcode primers. The specific sequences of the barcode linker and the barcode primer are not particularly limited as long as the genes can be ligated and enrichment of the DOCK3 gene is achieved. The composition of the present invention further comprises a reagent capable of indicating the status of ACTR3B gene in a biological sample, examples of which include, but are not limited to, additional barcode linkers and barcode primers, the specific sequences of which are not particularly limited as long as the gene can be ligated and the enrichment of ACTR3B gene is achieved. Preferably, the composition of the present invention comprises a reagent capable of simultaneously indicating the status of the DOCK3 and ACTR3B genes in a biological sample, and also preferably, the composition of the present invention consists of a reagent capable of simultaneously indicating the status of the DOCK3 and ACTR3B genes in a biological sample.
The status of the gene includes, but is not limited to, an increase or decrease in the expression level of the target gene (DOCK3 gene, ACTR3B), the presence or absence of insertion of a foreign gene into the target gene. A foreign gene refers to a gene derived from outside the organism of interest or study. In particular embodiments, the exogenous gene refers to an HPV gene, and examples of HPV genotyping include, but are not limited to: HPV genes related to common warts, flat warts, plantar warts, etc. such as HPV1, 2, 3, 4, 7, 10, 12, 15, etc.; HPV5, 8, 14, 17, 20, 36, 38 and other HPV genes related to epidermodysplasia verruciformis, vulvar cancer, penile cancer, anal cancer, prostatic cancer and bladder cancer; HPV-6, 11, 13, 32, 34, 40, 42, 43, 44, 54 and other HPV genes related to genital, anal, oropharyngeal and esophageal mucosa infection; HPV-16, 18, 30, 31, 33, 35, 53, 39 and the like related to cervical cancer, rectal cancer, oral cancer, tonsil cancer. HPV genes further include HPV32, 52, 56, 58, 68.
The gene insertion site is not particularly limited, and may be a site present within or near the gene of interest, and may be any site of any component/element thereof within or near the gene of interest, for example, any site including, but not limited to, upstream or downstream of a promoter, coding sequence, terminator, enhancer thereof.
Detection method
Step 1. sequencing a subject sample
First, a subject sample genomic DNA is sequenced in order to obtain a sequencing result. The type of the sample to be tested is not particularly limited, and includes, but is not limited to, TCT sample of cervical inflammation or hyperplasia, TCT sample of cervical lesion CIN or TCT sample of cervical cancer, or blood or tissue sample of the above-mentioned patient. Wherein, the TCT sample refers to a sample obtained according to a standard liquid-based thin-layer cell detection method. According to an embodiment of the present invention, before sequencing the genomic DNA of the sample infected with HPV in the subject, the method may further comprise: extracting genomic DNA from the subject sample. The extraction method is not particularly limited, and extraction may be performed by using a genomic DNA extraction method or an extraction kit known in the art.
According to an embodiment of the present invention, the subject genomic DNA is subjected to a sequencing step using nanopore sequencing technology (Nanorpore). Therefore, real-time monitoring of the sequencing process can be realized, and the target sequence is enriched, so that the sequencing time is obviously reduced. In addition, based on the technology, longer sequence reads (reads) can be obtained, the result of structural variation of a longer fragment is more reliable than that of millions of reads measured by an NGS platform, and meanwhile, the problem of sequence splicing caused by short sequences is avoided. In sequencing single DNA or RNA molecules, PCR amplification or chemical labeling of the sample is not required. Thus, a relatively low cost sequencing method for genotyping, high test mobility and rapid sample handling is provided. The instrument or sequencing platform for sequencing is not particularly limited, and examples of the instrument or platform for Nanopore sequencing include, but are not limited to, MinION sequencer, Gridios X5, Promethion, Flongle, by Oxford Nanopore Technologies. According to the library building and sequencing requirements of the nanopore sequencing technology platform, a sequencing library of the genome DNA of the sample of the subject is built, and then the sequencing library is sequenced so as to obtain a sequencing result.
Step 2. determining DNA fragment containing both HPV integration sequence and human genome sequence
Based on the above sequencing results, a DNA fragment containing both HPV sequences and human genome sequences was determined. Wherein, determining the DNA fragment comprising both HPV sequences and human genome sequences further comprises: filtering the sequencing result to obtain a filtered sequencing result; the filtered sequencing results are aligned with reference sequences to determine DNA fragments comprising both HPV integration sequences and human genome sequences. Wherein the reference sequence is human genome and HPV genome sequence.
It should be noted that, in the detection step of the present invention, the library creation includes a step of performing hybridization capture using a probe or a probe set, and the specific sequence of the probe or probe set used for the hybridization capture is not particularly limited as long as enrichment of HPV genome can be achieved. One skilled in the art can design such probes or probe sets based on the entire genome of the virus.
As used herein, unless otherwise indicated, the reference sequence is the human genomic data (hg18) downloaded (http:// genome. UCSC. edu /) from the UCSC database, including genes and duplicate annotations. COSMIC v58 and a series of oncogenes were downloaded from http:// www.sanger.ac.uk/genetics/CGP/Census. HPV genomic sequences were downloaded from http:// HPV-web. The dbSNP132 and thousand human genome project data that may be used herein are downloaded from the NCBI FTP website (http:// www.ncbi.nlm.nih.gov/Ftp /). Human disease-associated viral integration site data were obtained from http:// www.scbit.org/dbmi/drvis.
3. Authentication
The invention further includes validation of the sequencing data, which validated the reliability of HPV integration using sanger sequencing.
In the present invention, specific genes for diagnosis are obtained based on machine learning using HPV insertion data as characteristic data and disease progression as a label. Preferably, the machine learning is a random forest algorithm. Machine learning based analysis steps are known in the art.
Reagent kit
The invention further provides a kit comprising a composition as described herein. In addition to the above components, the kits of the present invention may include precautions related to the regulatory manufacture, use or sale of the diagnostic kit in a form prescribed by a governmental agency. In addition, the kits of the invention may be provided with detailed instructions for use, storage, and troubleshooting. The kit may optionally also be provided in a suitable device, preferably for robotic handling in a high throughput setting.
In certain embodiments, the components (e.g., oligonucleotides) of the kits of the invention may be provided as dry powders. When the reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent may also be disposed in another container. The container will typically comprise at least one vial, test tube, flask, bottle, syringe, and/or other container means, optionally in which the solvent is placed in equal portions. The kit may further comprise means for a second container comprising a sterile, pharmaceutically acceptable buffer and/or other solvent.
In certain embodiments, the components of the kits of the invention may be provided in the form of a solution, e.g., an aqueous solution. The concentrations or amounts of these ingredients, when present in aqueous solution, are readily determinable by those skilled in the art according to the various requirements. For example, for storage purposes, for example, the concentration of the oligonucleotide may be present in a higher form, and when in the working state or in use, the concentration may be reduced to the working concentration, for example, by diluting the higher concentration solution.
Where more than one component is present in a kit, the kit will also typically comprise a second, third or other additional container into which additional components may be separately placed. In addition, combinations of various components may be included in the container. Any of the compositions or reagents described herein can be a component of a kit.
The scheme of the invention will be explained with reference to the following examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruke et al, Huang Petang et al) or according to product instructions. The reagents or apparatus used are not indicated by the manufacturer, but are conventional products available commercially, for example from Illumina.
Example 1
Firstly, sample information:
1. the clinical sample information of the present invention is as follows: 16 TCT samples of HPV high-risk infection of early cervical inflammation or hyperplasia, 20 TCT or fresh tissue samples of grade CIN I, CIN II and CIN III cervical lesions, and 10 fresh tissue samples of cervical cancer.
And performing quality inspection on the sample after genome DNA extraction, and grouping the sample after the quality inspection is qualified.
2. Group entry criteria
The study may be selected for entry when the patient meets all of the following criteria:
1) patients who were examined for cervical cancer in my hospital;
2) HPV detection of high risk patients;
3) patients with abnormal colposcopy, TCT examination, cervical biopsy results.
4) Follow-up can be performed regularly.
5) The patient or family member was able to understand the study protocol and was willing to participate in the study, providing written informed consent.
3. Exclusion criteria
Patients cannot be selected for clinical study when any of the following conditions exist:
1) all examinations of cervix are normal
2) HPV detection negative
3) Pathologically confirmed as cervical neuroendocrine cancer;
4) combined with other malignant tumors
5) The follow-up visit is not standard or the clinical data is not complete.
6) Patients or family members were unable to coordinate the study or provide written informed consent.
Secondly, sequencing by taking tissue DNA as a sample
The gDNA of the TCT samples used in the test was cleaved at a length of 1kb or more. And (3) carrying out fragment sorting of more than 1kb on the broken DNA sample, and constructing a library on the broken product by using a three-generation nanopore library construction method.
Carrying out hybridization capture on HPV related probes of the third generation library, enriching HPV sequence information and carrying out nanopore sequencing. And carrying out QC quality control on the third-generation off-line data, and filtering out qualified sequences. Performing Blast comparison on the qualified sequences to find HPV integrated sequences containing human genome sequences and virus sequences. And (4) performing gene position annotation on the integrated sequence, sorting high-frequency genes and annotating genes related to the cervical cancer. And carrying out sanger sequencing verification on the detected high-frequency integration genes.
The method specifically comprises the following steps:
1. enrichment of target region
1) 1 mu g of tissue gDNA is taken, 1K to 3K is broken by Covarism220, the tissue gDNA is mixed with 5 mu g of human cot-1DNA, the mixture is dried by distillation at 60 ℃ by using a vacuum suction filter pump, then re-dissolved in optimized hybridization solution (containing 10 XSSPE, 10 XDenhart solution and 0.3% SDS), the mixture is incubated for 10min at room temperature and then put into a PCR instrument, a mixed 120nt ssDNA probe is added after 10min at 95 ℃, the temperature is slowly reduced to the hybridization temperature by reducing 0.1 ℃ per minute, and then the mixture is placed at 65 ℃ for hybridization for 4 to 16 hours.
2) Mixing the product obtained in the step 1) with streptavidin magnetic beads, incubating for 45min on a PCR instrument, and subsequently cleaning the magnetic beads with a cleaning solution.
3) And (3) enriching the product obtained in the step 2) by using a random primer, dNTP, Klenow exo-and the like, and performing quality control by using the Qubit4.0 and Agilent2100 capillary electrophoresis after purifying the product by using Agencour AMPure XP magnetic beads.
2. Building warehouse
1) Taking 100ng of each sample DNA and the enriched DNA library obtained in the step 1), constructing the library by using a PCR coding Kit of Oxford nanopore technologies, and repairing the End by using NEB Ultra II End-prep reaction buffer and Ultra II End-prep enzyme mix;
2) ligating Barcode Adapters to the product of step 1) using Blunt/TALigase Master Mix;
3) and (3) enriching the product obtained in the step 2) by using Long Amp Taq 2x Master Mix and Barcode Primers, purifying the product by using Agencour AMpure XP magnetic beads, and performing quality control by using Qubit4.0 and Agilent2100 capillary electrophoresis.
4) Libraries of different barcodes were pooled together for on-machine sequencing.
Third, the results
The third generation nanopore technology has the advantages of long reading time, rapidness, convenience and the like, and can be well adapted to clinical development and application. In order to search for CIN specific HPV integration sites, 15 HPV high-risk type probes are designed based on the whole virus genome, and based on a three-generation nanopore sequencing platform, the cervical exfoliated cell samples of 20-position entry subjects are captured and detected by probe hybridization. Wherein 1351 genes detected in the cervicitis group are related to HPV gene insertion, 4948 genes detected in the CIN I group are related to HPV gene insertion, 5562 genes detected in the CIN II group are related to HPV gene insertion, and 4041 genes detected in the CIN III group are related to HPV gene insertion. The results of the gene detection and clinical information are shown in Table 1.
TABLE 1
Compared with the NGS pre-sequencing result, 322 new integration sites are obtained based on the gene data obtained by the three generations of nanopore sequencing platforms. The reliability of the newly found 322 integration positions was verified by sanger sequencing.
The database is established by taking the insertion data of the 15902 genes in total as characteristics and taking different lesions of the cervix as labels. And (3) carrying out classification training on the database by using a random forest algorithm, and selecting the genes with the highest weight from the database, wherein the genes are DOCK3 genes and ACTR3B genes, and the two genes are new integration sites. Next, the DOCK3 genes were used as inflammation group-specific genes, respectively, and ACTR3B was classified as a specific gene of CIN group (including I, II and III).
Example 2
This example was used to analyze the expression of the DOCK3 gene and ACTR3B gene in different cervical tissues.
First, experiment method
Specific primers for the DOCK3 gene and ACTR3B gene were designed based on known public information, and primers were artificially synthesized by primer synthesis companies.
1. Total RNA of cervical secretions was extracted and tested for concentration, purity and integrity.
2. Carrying out a reverse transcription reaction:
1. mu.g of the extracted total RNA was used as a template, and the following reaction system was constructed and placed on ice to carry out a reaction.
The reverse transcription reaction system was then constructed:
the reverse transcription reaction system was reacted at 37 ℃ for 15 minutes.
And carrying out SYBRGreen qPCR detection, and drawing a melting curve. And detecting the expression conditions of the DOCK3 gene and the ACTR3B gene in the tissues.
Second, experimental results
In cervical secretions of healthy people, active expression of DOCK3 and ACTR3B was detected, and expression of the corresponding mRNA was present. However, in the inflammation group, the mRNA amount of DOCK3 was significantly reduced, but the mRNA amount in the CIN group sample was comparable to that of the healthy control population (fig. 1). In CIN group, the expression of ACTR3B gene was significantly reduced, but the amount of DOCK3 mRNA was not significantly reduced (FIG. 2).
The results showed that the insertion of HPV into the DOCK3 gene and the ACTR3B gene blocked the expression of the corresponding genes, and that the insertion of HPV gene into and around the DOCK3 gene is a phenomenon peculiar to the inflammatory group. The insertion of the HPV gene into and around the ACTR3B gene is a phenomenon specific to patients in the CIN group.
The research of the invention provides an effective means for further diagnosing or identifying cervical lesions, particularly distinguishing cervical inflammation from cervical CIN.
While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Many modifications and variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.
Claims (2)
1. Use of an agent in the preparation of a composition for use in differentiating cervicitis from cervical intraepithelial neoplasia detection by a method comprising the steps of, wherein the agent comprises an agent capable of indicating the status of ACTR3B and DOCK3 genes in a biological sample, the method comprising:
a. collecting a biological sample from a subject, and separating DNA to obtain a DNA fragment with the length of 1-3 Kb;
b. separating a target sequence from the DNA fragment, constructing a library, connecting a bar code joint at the tail end, and enriching by adopting a bar code primer to enable the target sequence or part of the target sequence to pass through a nanopore located in a chip, wherein the chip is arranged near an electrode, the electrode can detect current passing through the nanopore, and information of the target sequence is detected through current change;
diagnosing the subject as having or having a high risk of having cervical intraepithelial neoplasia when the presence of HPV gene insertion is detected within or near the ACTR3B gene; when the presence of HPV gene insertion is detected within or near the DOCK3 gene, the subject is diagnosed as a cervicitis patient.
2. The use of claim 1, wherein the enrichment comprises mixing a DNA fragment with a length of 1-3Kb with human cot-1DNA, evaporating to dryness, redissolving in an optimized hybridization solution, incubating at room temperature, maintaining at 93-97 ℃ for 5-15 minutes, adding a probe set consisting of a plurality of probes, slowly cooling to the hybridization temperature by lowering the temperature by 0.1 ℃ per minute to improve the specificity of the captured target sequence and the binding stability of the long fragment, hybridizing at 63-67 ℃ for 4-16 hours, mixing the product with streptavidin magnetic beads, incubating for 45 minutes, washing the magnetic beads with a washing solution, and synthesizing and enriching using a random primer, dNTP, an enzyme having a single-stranded DNA as a guide for duplex synthesis activity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111594414.8A CN114250302B (en) | 2021-12-23 | 2021-12-23 | Composition and kit for detecting cervical intraepithelial neoplasia and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111594414.8A CN114250302B (en) | 2021-12-23 | 2021-12-23 | Composition and kit for detecting cervical intraepithelial neoplasia and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114250302A CN114250302A (en) | 2022-03-29 |
| CN114250302B true CN114250302B (en) | 2022-08-19 |
Family
ID=80794835
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111594414.8A Active CN114250302B (en) | 2021-12-23 | 2021-12-23 | Composition and kit for detecting cervical intraepithelial neoplasia and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114250302B (en) |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2695814A1 (en) * | 2007-09-07 | 2009-04-23 | Universite Libre De Bruxelles | Methods and tools for prognosis of cancer in her2+ patients |
| US20170058351A1 (en) * | 2013-11-28 | 2017-03-02 | Stichting Het Nederlands Kanker Instituut-Antoni van Leeuwenhoek Ziekenhuis | Methods for Molecular Classification of BRCA-Like Breast and/or Ovarian Cancer |
| WO2019232485A1 (en) * | 2018-05-31 | 2019-12-05 | Nvigen, Inc. | Accurate blood test to predict cancer incidence, recurrence, guide and monitor treatment intervention |
| CN110499364A (en) * | 2019-07-30 | 2019-11-26 | 北京凯昂医学诊断技术有限公司 | A kind of probe groups and its kit and application for detecting the full exon of extended pattern hereditary disease |
-
2021
- 2021-12-23 CN CN202111594414.8A patent/CN114250302B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN114250302A (en) | 2022-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2021128519A1 (en) | Combination of dna methylation biomarkers, and detection method therefor and kit thereof | |
| TW202340477A (en) | Methods and systems for tumor detection | |
| EP3800273A1 (en) | Use of detection reagent for detecting methylation of genes associated with colorectal cancer, and kit | |
| US20150361502A1 (en) | Method for screening cancer | |
| JP6606554B2 (en) | Use of the methylated site of the Y chromosome as a diagnostic marker for prostate cancer | |
| WO2019076018A1 (en) | Method for constructing amplicon library for detecting low-frequency mutation of target gene | |
| CN111424093B (en) | Kit, device and method for lung cancer diagnosis | |
| CN109593847B (en) | Primer pair, kit and method for detecting stability of NR24 locus of microsatellite | |
| CN110317877A (en) | Application of the unstable variation of one group chromosome in preparation diagnosis bladder transitional cell carcinoma, the reagent or kit of assessing prognosis | |
| CN113215260A (en) | Application of GSTP1, APC and RASSF1 in preparation of prostate cancer markers and kit thereof | |
| CN111455053B (en) | Exosome RNA molecular marker combination for colorectal adenoma diagnosis and application thereof | |
| CN114250302B (en) | Composition and kit for detecting cervical intraepithelial neoplasia and application | |
| CN114196755B (en) | Composition for diagnosing cervical lesions in a subject and use thereof | |
| CN111020710A (en) | ctDNA high-throughput detection of hematopoietic and lymphoid tissue tumors | |
| CN113817822B (en) | Tumor diagnosis kit based on methylation detection and application thereof | |
| CN111349700B (en) | Gene marker combination, kit and method for detecting urothelial cancer | |
| CN114107514A (en) | miRNA molecular marker for colorectal cancer diagnosis and kit thereof | |
| CN114921558B (en) | Application of hsa _ circ _0044235 level in serum as breast cancer diagnostic marker | |
| WO2024183797A1 (en) | Use of plasma cell-free dna methylation marker in liver tumor detection | |
| CN116121453B (en) | Improved structure of high-sensitivity fluorescent PCR detection primer and probe and application thereof | |
| CN113005199B (en) | Primer composition for detecting microsatellite instability, kit and application | |
| TWI417546B (en) | Dna methylation biomarkers for prognosis prediction of lung adenocarcinoma | |
| CN116396960A (en) | Nucleic acid molecules and kits for detecting cervical intraepithelial neoplasia and cervical squamous cell carcinoma | |
| CN114395623A (en) | Gene methylation detection primer composition and kit and application thereof | |
| CN116179693A (en) | Application of reagent for detecting methylation level of target region in preparation of gynecological malignant tumor 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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |