CN116287263A - Composition and kit for thyroid nodule detection - Google Patents
Composition and kit for thyroid nodule detection Download PDFInfo
- Publication number
- CN116287263A CN116287263A CN202310194462.0A CN202310194462A CN116287263A CN 116287263 A CN116287263 A CN 116287263A CN 202310194462 A CN202310194462 A CN 202310194462A CN 116287263 A CN116287263 A CN 116287263A
- Authority
- CN
- China
- Prior art keywords
- detection
- seq
- methylation
- gene
- thyroid
- 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
- 238000001514 detection method Methods 0.000 title claims abstract description 69
- 208000009453 Thyroid Nodule Diseases 0.000 title claims abstract description 43
- 208000024770 Thyroid neoplasm Diseases 0.000 title claims abstract description 38
- 239000000203 mixture Substances 0.000 title claims abstract description 26
- 230000011987 methylation Effects 0.000 claims abstract description 59
- 238000007069 methylation reaction Methods 0.000 claims abstract description 59
- 239000000523 sample Substances 0.000 claims abstract description 38
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 16
- 150000007523 nucleic acids Chemical class 0.000 claims description 23
- 101150038861 Uchl1 gene Proteins 0.000 claims description 11
- 108020004707 nucleic acids Proteins 0.000 claims description 11
- 102000039446 nucleic acids Human genes 0.000 claims description 11
- 101150077103 TPO gene Proteins 0.000 claims description 8
- 108091008146 restriction endonucleases Proteins 0.000 claims description 8
- 101150072693 KIF1A gene Proteins 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 7
- 230000003321 amplification Effects 0.000 claims description 7
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 239000003623 enhancer Substances 0.000 claims description 4
- 229910001425 magnesium ion Inorganic materials 0.000 claims description 4
- 238000012163 sequencing technique Methods 0.000 claims description 3
- 238000004949 mass spectrometry Methods 0.000 claims description 2
- 230000003211 malignant effect Effects 0.000 abstract description 29
- 108090000623 proteins and genes Proteins 0.000 abstract description 25
- 101710113649 Thyroid peroxidase Proteins 0.000 abstract description 21
- 101000971638 Homo sapiens Kinesin-like protein KIF1A Proteins 0.000 abstract description 20
- 108010005656 Ubiquitin Thiolesterase Proteins 0.000 abstract description 13
- 238000003753 real-time PCR Methods 0.000 abstract description 5
- 210000001685 thyroid gland Anatomy 0.000 description 21
- 206010054107 Nodule Diseases 0.000 description 20
- 102100027188 Thyroid peroxidase Human genes 0.000 description 17
- 102100021527 Kinesin-like protein KIF1A Human genes 0.000 description 16
- 238000000034 method Methods 0.000 description 14
- 108091028043 Nucleic acid sequence Proteins 0.000 description 12
- 239000003550 marker Substances 0.000 description 11
- 238000011144 upstream manufacturing Methods 0.000 description 10
- 102000005918 Ubiquitin Thiolesterase Human genes 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 230000035945 sensitivity Effects 0.000 description 7
- 201000002510 thyroid cancer Diseases 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 5
- 230000007067 DNA methylation Effects 0.000 description 4
- 101100427383 Dictyostelium discoideum uch1 gene Proteins 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 206010004412 Benign neoplasm of thyroid gland Diseases 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 238000013211 curve analysis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 238000007477 logistic regression Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 description 1
- 208000006274 Brain Stem Neoplasms Diseases 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 208000031448 Genomic Instability Diseases 0.000 description 1
- 108700019961 Neoplasm Genes Proteins 0.000 description 1
- 102000048850 Neoplasm Genes Human genes 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000023715 cellular developmental process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000002380 cytological effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000008995 epigenetic change Effects 0.000 description 1
- 230000004049 epigenetic modification Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 230000006607 hypermethylation Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 208000003154 papilloma Diseases 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000012175 pyrosequencing Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 238000002604 ultrasonography Methods 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a composition and a kit for thyroid nodule detection, wherein the composition comprises a detection reagent for detecting methylation levels of KIF1A genes, TPO genes and UCHL1 genes. According to the invention, the target genes of the primer probe groups are different markers after methylation of the thyroid nodule related genes, so that combination of MSRE and fluorescent quantitative PCR is realized, methylation detection of the thyroid nodule related genes is realized, and effective judgment of benign and malignant thyroid nodule is further realized.
Description
Technical Field
The invention relates to the field of biotechnology and medicine, in particular to a composition and a kit for thyroid nodule detection.
Background
Thyroid nodule is a lump formed in thyroid tissues after abnormal proliferation of thyroid cells, the new incidence number of thyroid cancer in China is 20.1 ten thousand per year, and the thyroid nodule is malignant tumor with the highest incidence rate. Thyroid cancer includes at least four different types of thyroid malignancy: papillomas, follicular tumors, medullary tumors, and undifferentiated tumors. Thyroid cancer is highly treatable, and most patients have a good prognosis after thyroidectomy. Thyroid nodules can be classified as benign and malignant, but some benign and malignant thyroid nodules have no obvious difference in cytologic characteristics, resulting in unnecessary excision of the thyroid gland in patients without cancer. The data show that in the united states, 5 tens of thousands of thyroid nodule patients undergo unnecessary thyroidectomy due to diagnostic errors each year. Therefore, the thyroid nodule benign and malignant can be accurately judged, unnecessary operations can be reduced, more accurate treatment is realized, and the burden of a patient is reduced.
Currently, assessment of thyroid nodules is mainly performed by ultrasonic examination and fine needle aspiration biopsy (Fine needle aspiration biopsy, FNAB), high-resolution ultrasound is the first examination of thyroid nodules, and ultrasonic examination of nodules with malignant signs is combined with FNAB detection to determine the nature of the nodules. In the cytological examination result, up to 20% of the nodules are uncertain thyroid nodules, and molecular detection of fine needle puncture samples of the thyroid nodules with uncertain benign and malignant characteristics can help to improve the accuracy of identifying the benign and malignant characteristics of the thyroid nodules before operation of a clinician, so that excessive operation and missed diagnosis of the malignant nodules are avoided, and the positive predictive value (positive predictive value, PPV) of the existing thyroid cancer gene detection products is very low (42% -77%), so that a more accurate molecular diagnosis tool is needed.
Thyroid carcinogenesis has multiple mechanisms involved and epigenetic changes play an important role in thyroid cancer. DNA methylation is an important epigenetic modification that plays a critical role in normal cellular development, gene expression patterns, and genomic stability, an abnormality in function that is one of the earliest molecular changes occurring in the course of cancer, and is tissue-specific, abnormal DNA methylation usually occurs in the promoter region of transcription factors, leading to tumor development primarily through hypermethylation or hypomethylation. In clinical applications, DNA methylation has potential as a diagnostic screening marker and therapeutic target.
Disclosure of Invention
The invention provides a composition and a kit for thyroid nodule detection, which are used for realizing effective detection of malignant thyroid nodules.
The invention provides a composition for thyroid nodule detection, which comprises detection reagents for detecting methylation levels of KIF1A genes, TPO genes and UCHL1 genes.
Further, the composition comprises detection reagents for detecting the methylation level of:
a region of the KIF1A gene shown as SEQ ID No. 13;
the region of TPO gene shown in SEQ ID No. 14;
the UCHL1 gene is shown as a region shown as SEQ ID No. 15.
Specifically, the region of SEQ ID NO:13 of the KIF1A (Genbank accession number: NG_ 029724.1) gene is shown below:
AGCTGTCTAGCTGGTTTCGGCGGGGCTGGGGCTGGGAGTGGGGGGCCCGTGTCTTGC AGAAGTGGATGTGAATCTCCCCCAGCCACCTGCAAGGCGCTCGCACGCAGCTGTCGGGAC TGAGGTGAAGCCGG；
the SEQ ID NO:14 region of the TPO (Genbank accession number: NG_ 011581.2) gene is shown below:
AAAACAAGTCCACTGCCGCCCCTCCCACAGTCTCTCCGCTCTCCCTGCATCAGCCAGT GGTTCACACTGAGGACACACAGGTGAATCACGTTGAGAATTCCCAGCAGAACCGTAGTTTT CT；
the SEQ ID NO:15 region of the UCHL1 (Genbank accession number: NG_ 012931.1) gene is shown below:
AATTAGCCGGGTGTGGTGGCGGGCGCCTGTAGTCCCAGCTACTCCGGAGGCTGAGGCA GGAGAATGGCGTGAACCGGGGAGGCGGAGCTTGCAGTGAGCCGAGATCGCGCCACT。
further, the detection reagent is a detection reagent used in nucleic acid amplification, nucleic acid sequencing or nucleic acid mass spectrometry to detect methylation levels.
Further, the detection reagent is a primer and/or a probe.
Further, at least one of the primers of the primer-probe set comprises an upstream primer, a downstream primer and a probe respectively having sequences of SEQ ID No.1 to SEQ ID No. 3.
Further, at least one of the primers of the primer-probe set comprises an upstream primer, a downstream primer and a probe respectively having sequences of SEQ ID No.4 to SEQ ID No. 6.
Further, at least one of the primers of the primer-probe set comprises an upstream primer, a downstream primer and a probe respectively having sequences of SEQ ID No.7 to SEQ ID No. 9.
Further, the molar concentration ratio of the primers and/or probes corresponding to each two different genes is 1-10.
The invention also discloses a detection kit, which comprises the composition.
Further, the detection kit also comprises methylation sensitive restriction enzyme mix.
Further, the detection kit also comprises a hot start enzyme and a GC enhancer
Further, the detection kit also comprises dNTPs, PCR amplification buffer and Mg ions.
Compared with the prior art, the method has the advantages that the combination of MSRE and fluorescent quantitative PCR is realized by setting the target genes of the primer probe groups as different markers after the methylation of the thyroid nodule related genes, so that the methylation detection of the thyroid nodule related genes is realized, and further, the effective judgment of benign and malignant thyroid nodule is realized.
Drawings
FIG. 1 shows the methylation levels of KIF1A genes in malignant and benign thyroid nodule tissues according to example 1 of the present invention;
FIG. 2 shows the methylation levels of TPO genes in malignant and benign tissues of thyroid nodule in example 1 of the present invention;
FIG. 3 shows the methylation levels of UCHL1 genes of malignant and benign thyroid nodule tissues according to example 1 of the present invention;
FIG. 4 is an amplification curve of the marker and internal standard sample according to example 2 of the present invention;
FIG. 5 is a sample amplification curve without methylation of the target gene according to example 2 of the present invention;
FIG. 6 is a ROC analysis of KIF1A according to an embodiment of the invention;
FIG. 7 is a chart of ROC analysis of an embodiment TPO of the invention;
FIG. 8 is a ROC analysis chart of UCHL1 of an embodiment of the invention;
FIG. 9 is a ROC analysis of KIF1A, TPO, UCHL1 according to the invention.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution of the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments.
The DNA methylation detection method has a plurality of methods, such as a pyrosequencing method, a methylation sensitivity high-resolution dissolution curve method, a methyl light fluorescence PCR method, a methylation specificity PCR method, a methylation sensitivity restriction enzyme Method (MSRE) and the like, and compared with other detection methods, the MSRE method does not need the transformation of sulfite, and has the advantages of high sensitivity, simplicity in operation, short time consumption and the like. The embodiment of the invention adopts MSRE combined with fluorescent PCR technology to realize methylation detection of genes.
The embodiment of the invention discloses a composition for thyroid nodule detection, which comprises a detection reagent for detecting methylation levels of KIF1A genes, TPO genes and UCHL1 genes.
Optionally, the composition comprises detection reagents for detecting the methylation level of:
a region of the KIF1A gene shown as SEQ ID No. 13;
the region of TPO gene shown in SEQ ID No. 14;
the UCHL1 gene is shown as a region shown as SEQ ID No. 15.
Specifically, the region of SEQ ID NO:13 of the KIF1A (Genbank accession number: NG_ 029724.1) gene is shown below:
AGCTGTCTAGCTGGTTTCGGCGGGGCTGGGGCTGGGAGTGGGGGGCCCGTGTCTTGC AGAAGTGGATGTGAATCTCCCCCAGCCACCTGCAAGGCGCTCGCACGCAGCTGTCGGGAC TGAGGTGAAGCCGG；
the SEQ ID NO:14 region of the TPO (Genbank accession number: NG_ 011581.2) gene is shown below:
AAAACAAGTCCACTGCCGCCCCTCCCACAGTCTCTCCGCTCTCCCTGCATCAGCCAGT GGTTCACACTGAGGACACACAGGTGAATCACGTTGAGAATTCCCAGCAGAACCGTAGTTTT CT；
the SEQ ID NO:15 region of the UCHL1 (Genbank accession number: NG_ 012931.1) gene is shown below:
AATTAGCCGGGTGTGGTGGCGGGCGCCTGTAGTCCCAGCTACTCCGGAGGCTGAGGCA GGAGAATGGCGTGAACCGGGGAGGCGGAGCTTGCAGTGAGCCGAGATCGCGCCACT。
here, it was experimentally analyzed (see in particular example 1 of the present invention) that KIF1A, TPO, UCHL and the like are methylated at a relatively high rate in thyroid cancer.
The markers of the embodiment of the invention comprise a KIF1A gene methylation marker, a TPO gene methylation marker and a UCHL1 gene methylation marker, and by adopting the three markers, the malignant tissue of the thyroid nodule can be specifically analyzed and identified.
Optionally, at least one of the primers of the primer-probe set comprises an upstream primer, a downstream primer and a probe respectively comprising sequences of SEQ ID No.1 to SEQ ID No. 3.
Optionally, at least one of the primers of the primer-probe set comprises an upstream primer, a downstream primer and a probe respectively comprising sequences of SEQ ID No.4 to SEQ ID No. 6.
Optionally, at least one of the primers of the primer-probe set comprises an upstream primer, a downstream primer and a probe respectively comprising sequences of SEQ ID No.7 to SEQ ID No. 9.
Wherein, the sequences of SEQ ID No.1 to SEQ ID No.9 of the examples of the present invention are shown in the following table.
The 4 fluorescence channels adopted in the embodiment of the invention are FAM, HEX, CY and ROX channels respectively, but the application is not limited to the above, and any other combination of fluorescence channels can be adopted; meanwhile, different targets should correspond to different fluorescent channels, so that workers can conveniently obtain amplification results of different gene methylation.
Further, the 3' end of the probe has a quenching group, such as BHQ1, BHQ2.
Alternatively, the molar concentration ratio of the primers and/or probes for each two different genes is 1-10.
Wherein, the molar concentration ratio of the primers and/or probes corresponding to each two different genes in the embodiment of the invention is 1.
In another aspect, the embodiment of the invention also discloses a detection kit, which comprises the composition.
Optionally, the detection kit further comprises a methylation sensitive restriction enzyme mix.
Alternatively, the methylation sensitive restriction enzyme mix includes HpyCH4IV, acil and HpaII.
The methylation detection of the promoter region of the KIF1A, TPO, UCHL gene is carried out by combining an enzyme digestion method with qPCR by adopting methylation sensitive restriction enzyme mix. The detection sensitivity of the product is 90%, the specificity is 92%, the product is superior to serological examination, fine needle cell puncture detection and molecular diagnosis products which are on the market at present, and the detection specificity of the product is better.
Optionally, the detection kit further comprises a hot start enzyme and a GC enhancer
Optionally, the detection kit further comprises dNTPs, PCR amplification buffer and Mg ions.
Based on the knowledge of the conventional fluorescent quantitative PCR technology, the person skilled in the art is motivated to select the dosage of PCR amplification buffer, mg ions, dNTPs, methylation sensitive restriction enzyme, hot start enzyme and GC enhancer by himself, so that the embodiment of the invention is not limited by specific dosage.
The detection kit provided by the embodiment of the invention provides a novel molecular diagnosis technical path by detecting methylation markers in DNA of thyroid nodule samples, can directly take thyroid nodule tissues and patient plasma as samples, extracts nucleic acid from the tissue samples and the patient plasma, and judges benign and malignant thyroid nodule through multi-target methylation combined detection.
To specifically illustrate the solution and effect of the embodiments of the present invention, embodiments 1 to 3 are now disclosed.
Example 1: gene methylation expression level detection
step 2, extracting nucleic acid from the thyroid nodule tissue sample by adopting a St.Hunan organism S1008 nucleic acid extraction kit, measuring the concentration, diluting a proper amount of sample to 1 ng/. Mu.l, and then detecting according to a detection flow;
Methylation level = 2 Delta Ct sample to be tested X 100%, Δct=ct target gene-Ct reference gene. As shown in fig. 1-3, three targets of the invention are highly methylated in thyroid malignant nodule tissue, while the methylation level of thyroid benign nodules is low, which indicates that the methylation level of the three targets is highly correlated with cancer tissue and can be used as a detection marker of thyroid cancer in thyroid nodules.
Example 2: reference detection
The sequence of the primer probe set adopted by the embodiment 2 of the invention aiming at the methylation marker of the KIF1A, TPO, UCHL1 gene and the internal standard gene (action) is shown in the following table:
the embodiment 2 of the invention is designed to detect the sequence shown in SEQ ID NO.1 of an upstream primer nucleic acid sequence and SEQ ID NO.2 of a downstream primer nucleic acid sequence of a KIF1A gene methylation marker and the sequence shown in SEQ ID NO.3 of a probe nucleic acid sequence. The primer is used for detecting the sequence of an upstream primer nucleic acid sequence shown as SEQ ID NO.4, the sequence of a downstream primer nucleic acid sequence shown as SEQ ID NO.5 and the sequence of a probe nucleic acid sequence shown as SEQ ID NO.6 of the TPO gene methylation marker. The primer is used for detecting the sequence of the UCHL1 gene methylation marker, the upstream primer nucleic acid sequence of which is shown as SEQ ID NO.7, the downstream primer nucleic acid sequence of which is shown as SEQ ID NO.8, and the probe nucleic acid sequence of which is shown as SEQ ID NO. 9. The invention of example 2 also uses ACTB as an internal standard. The upstream primer nucleic acid sequence of the internal standard ACTB is shown as a sequence shown as SEQ ID NO.10, the downstream primer nucleic acid sequence is shown as a sequence shown as SEQ ID NO.11, and the probe nucleic acid sequence is shown as a sequence shown as SEQ ID NO. 12.
The positive samples of example 2 of the present invention were: standard methylated human genomic DNA (KIF 1A, TPO, UCHL1 and internal standard ACTB) at a concentration of 1 ng/. Mu.l 10% methylated DNA;
the negative samples of example 2 of the present invention were: the sequencing shows that the concentration of the human genome DNA without the methylation of the KIF1A, TPO, UCHL1 gene is 2 ng/. Mu.l, and the concentration of the human genome DNA containing the internal standard ACTB genome DNA is 1 ng/. Mu.l.
Methylation sensitive restriction enzyme mix: including HpyCH4IV, acil and HpaII.
The process of the embodiment 2 of the invention is as follows:
step 2, adding a sample into a PCR reaction tube according to 10 mu L/reaction, sequentially adding 40 mu L of PCR reaction liquid, covering a PCR tube cover, shaking and mixing uniformly, and performing instantaneous centrifugation for 5s;
3.1. fluorescence detection channel selection: selecting ROX channel (report: ROX, quantiser: none) to detect UCHL1; selecting FAM channel (report: FAM, quantiser: none) to detect KIF1A; selecting HEX channel (report: HEX, quantiser: none) to detect TPO; CY5 channel (Reportere: CY5, quantiser: none) was selected as an internal standard to detect housekeeping gene ACTB;
3.2. the fluorescent quantitative PCR reaction conditions are as follows:
the amplification curves of the positive standard and the negative standard in the embodiment 2 of the invention are shown in fig. 4 and 5, and the PCR reaction liquid in the embodiment 2 of the invention can realize the specific amplification of the positive standard and has good sensitivity.
Example 3: clinical sample detection
The procedure of example 3 of the present invention is as follows:
step 2, extracting plasma cfDNA by adopting a Norflua plasma free DNA extraction kit;
Judging whether the experimental result meets the quality control requirement according to the Ct value of the internal standard gene action, if so, determining that>27, not including the result statistics; methylation levels of three target genes of KIF1A, TPO, UCHL1 were obtained from the methylation level calculation formula of example 1. The ROC curve analysis of KIF1A, TPO, UCHL1 is shown in FIG. 6, FIG. 7 and FIG. 8, respectively, and the PCR reaction solution and the PCR amplification detection process of the embodiment 3 of the invention can specifically detect the methylation marker of the KIF1A, TPO, UCHL gene. In detecting plasma samples from 100 patients with thyroid nodules (50 thyroid malignant nodules and 50 thyroid benign nodules), the methylation of the KIF1A gene was performed on thyroidThe area under the ROC curve for the differentiation of malignant and thyroid benign nodules was 0.838; the area under the ROC curve for the differentiation of the TPO gene methylation from thyroid malignant nodules and thyroid benign nodules was 0.822; the area under the ROC curve for the differentiation of UCHL1 gene methylation from thyroid malignant nodules and thyroid benign nodules was 0.820. ROC curve analysis for three-gene combined detection as shown in fig. 9, the area under the ROC curve for the three-gene combined detection to distinguish between a thyroid malignant nodule and a thyroid benign nodule was 0.952. Binary Logistic regression analysis was performed on the methylation levels detected in combination with genes, logistic regression equation = -7.3554+17.3639 x kif1a methylation level +17.4362 x tpo methylation level +18.624 x uchl1 methylation level. From the detection results of the fluorescent quantitative PCR, the methylation level of each target was calculated (methylation level=2 Delta Ct sample to be tested X 100%) and determining that the result is positive when the result is greater than 0 by using a logistics regression equation, namely malignant nodules, wherein 4 out of 50 thyroid benign nodules and 45 out of 50 thyroid malignant nodules are positive according to an interpretation standard, and the detection sensitivity of the PCR reaction solution (the kit component) of the embodiment of the invention is 90% and the specificity is 92%, so that the method for identifying benign and malignant thyroid nodules is provided for clinic by jointly detecting the methylation level of the promoter region of the KIF1A, TPO, UCHL1 gene.
In conclusion, the KIF1A, TPO, UCHL1 is a methylation gene specific to thyroid malignant nodules, and the sensitivity and the specificity of the combined detection of the KIF1A, TPO, UCHL1 gene on thyroid nodule plasma sample detection are higher than those of single-target detection, so that an effective and accurate method is provided for judging benign and malignant thyroid nodules. In addition, the components of the detection kit provided by the embodiment of the invention can be used for carrying out the enzyme digestion treatment and the PCR amplification of the nucleic acid sample in the same reaction tube, and the detection kit has the advantages of simplicity and convenience in operation.
Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made to the specific embodiments of the present invention after reading the present specification, and these modifications and variations do not depart from the scope of the invention as claimed in the pending claims.
Claims (10)
1. A composition for thyroid nodule detection, comprising a detection reagent for detecting methylation levels of KIF1A gene, TPO gene and UCHL1 gene.
2. The composition for thyroid nodule detection of claim 1, wherein the composition comprises detection reagents for detecting the methylation level of:
a region of the KIF1A gene shown as SEQ ID No. 13;
the region of TPO gene shown in SEQ ID No. 14;
the UCHL1 gene is shown as a region shown as SEQ ID No. 15.
3. The composition for thyroid nodule detection according to claim 2, wherein the detection reagent is a detection reagent used in nucleic acid amplification, nucleic acid sequencing or nucleic acid mass spectrometry to detect methylation levels.
4. A composition for thyroid nodule detection as claimed in claim 3 wherein said detection reagent is a primer and/or probe.
5. The composition for thyroid nodule detection according to claim 4, wherein said detection reagent comprises a primer and a probe as shown in SEQ ID nos. 1 to 9.
6. The composition for thyroid nodule detection according to claim 5, wherein said detection reagent further comprises a primer and a probe as set forth in SEQ ID No.10 to SEQ ID No. 12.
7. A test kit comprising the composition of any one of claims 1-6.
8. The test kit of claim 7, further comprising a methylation sensitive restriction enzyme mix.
9. The test kit of claim 7, further comprising a hot start enzyme, GC enhancer.
10. The detection kit according to claim 7, further comprising dNTPs, PCR amplification buffer, mg ions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310194462.0A CN116287263A (en) | 2023-03-02 | 2023-03-02 | Composition and kit for thyroid nodule detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310194462.0A CN116287263A (en) | 2023-03-02 | 2023-03-02 | Composition and kit for thyroid nodule detection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116287263A true CN116287263A (en) | 2023-06-23 |
Family
ID=86837267
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310194462.0A Pending CN116287263A (en) | 2023-03-02 | 2023-03-02 | Composition and kit for thyroid nodule detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116287263A (en) |
-
2023
- 2023-03-02 CN CN202310194462.0A patent/CN116287263A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6369857B2 (en) | Method for obtaining information on hepatocellular carcinoma, and marker and kit for obtaining information on hepatocellular carcinoma | |
| US20150361502A1 (en) | Method for screening cancer | |
| JP6269492B2 (en) | Method for obtaining information on hepatocellular carcinoma, and marker and kit for obtaining information on hepatocellular carcinoma | |
| KR102472253B1 (en) | Composition for diagnosing liver cancer using CpG methylation status of specific gene and uses thereof | |
| JP6395131B2 (en) | Method for acquiring information on lung cancer, and marker and kit for acquiring information on lung cancer | |
| JP6381020B2 (en) | Method for obtaining information on colorectal cancer, and marker and kit for obtaining information on colorectal cancer | |
| CN107630093B (en) | Reagent, kit, detection method and application for diagnosing liver cancer | |
| CN112410425A (en) | Gene methylation digital PCR kit for early screening of liver cancer and application thereof | |
| KR102472257B1 (en) | Composition for diagnosing colorectal cancer or adenoma using CpG methylation status of LINC01798 gene and uses thereof | |
| US11535897B2 (en) | Composite epigenetic biomarkers for accurate screening, diagnosis and prognosis of colorectal cancer | |
| CN116287263A (en) | Composition and kit for thyroid nodule detection | |
| CN116555423A (en) | Lung cancer methylation marker combination, detection product and application thereof | |
| US11542559B2 (en) | Methylation-based biomarkers in breast cancer screening, diagnosis, or prognosis | |
| CN109355362B (en) | High-sensitivity SNPs detection system and application | |
| JP6418594B2 (en) | Method for obtaining information on endometrial cancer, and marker and kit for obtaining information on endometrial cancer | |
| CN114908086B (en) | Primer probe composition, kit and detection method for detecting EGFR 19del based on digital PCR | |
| JP6636105B2 (en) | Method for obtaining information on colorectal cancer, and marker and kit for obtaining information on colorectal cancer | |
| JP6551656B2 (en) | Method for obtaining information on ovarian cancer, and marker for obtaining information on ovarian cancer and kit for detecting ovarian cancer | |
| CN116590419A (en) | Composition for detecting liver cancer, kit and application thereof | |
| CN117778573A (en) | Nucleic acid combination for detecting thyroid cancer biomarker, kit and application | |
| CN116555422A (en) | Lung cancer methylation marker, detection kit and application thereof | |
| CN118006769A (en) | Lung cancer related 7 gene methylation joint detection kit and use method thereof | |
| CN117106899A (en) | Primer pair, primer probe combination, kit and application for detecting benign and malignant lung nodule | |
| WO2023066972A1 (en) | Dna methylation signature for diagnosing hepatocellular carcinoma | |
| CN117402973A (en) | Nucleic acid reagent for detecting breast cancer, kit and application |
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 |