CN117402975A - Probe, kit and drop-off ddPCR method for quantitatively detecting IDH1 gene mutation - Google Patents
Probe, kit and drop-off ddPCR method for quantitatively detecting IDH1 gene mutation Download PDFInfo
- Publication number
- CN117402975A CN117402975A CN202311722602.3A CN202311722602A CN117402975A CN 117402975 A CN117402975 A CN 117402975A CN 202311722602 A CN202311722602 A CN 202311722602A CN 117402975 A CN117402975 A CN 117402975A
- Authority
- CN
- China
- Prior art keywords
- probe
- drop
- mutation
- idh1
- primer
- 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
- 239000000523 sample Substances 0.000 title claims abstract description 113
- 206010064571 Gene mutation Diseases 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 23
- 101150046722 idh1 gene Proteins 0.000 title claims description 44
- 230000035772 mutation Effects 0.000 claims abstract description 35
- 102100039905 Isocitrate dehydrogenase [NADP] cytoplasmic Human genes 0.000 claims description 28
- 101001042041 Bos taurus Isocitrate dehydrogenase [NAD] subunit beta, mitochondrial Proteins 0.000 claims description 27
- 101000960234 Homo sapiens Isocitrate dehydrogenase [NADP] cytoplasmic Proteins 0.000 claims description 27
- 238000006243 chemical reaction Methods 0.000 claims description 26
- 239000013612 plasmid Substances 0.000 claims description 21
- 239000002773 nucleotide Substances 0.000 claims description 16
- 125000003729 nucleotide group Chemical group 0.000 claims description 16
- 230000036438 mutation frequency Effects 0.000 claims description 15
- 102000004190 Enzymes Human genes 0.000 claims description 11
- 108090000790 Enzymes Proteins 0.000 claims description 11
- 238000010791 quenching Methods 0.000 claims description 10
- 230000000171 quenching effect Effects 0.000 claims description 10
- 238000012408 PCR amplification Methods 0.000 claims description 9
- 238000011304 droplet digital PCR Methods 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 7
- 239000012295 chemical reaction liquid Substances 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 238000001514 detection method Methods 0.000 abstract description 23
- 108090000623 proteins and genes Proteins 0.000 abstract description 13
- 230000000295 complement effect Effects 0.000 abstract description 6
- 239000007788 liquid Substances 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 description 7
- KPGXRSRHYNQIFN-UHFFFAOYSA-L 2-oxoglutarate(2-) Chemical compound [O-]C(=O)CCC(=O)C([O-])=O KPGXRSRHYNQIFN-UHFFFAOYSA-L 0.000 description 6
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 208000032612 Glial tumor Diseases 0.000 description 5
- 206010018338 Glioma Diseases 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- HWXBTNAVRSUOJR-UHFFFAOYSA-N 2-hydroxyglutaric acid Chemical compound OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 description 4
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 210000002824 peroxisome Anatomy 0.000 description 4
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000007847 digital PCR Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 238000007480 sanger sequencing Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 101710102690 Isocitrate dehydrogenase [NADP] cytoplasmic Proteins 0.000 description 2
- 101710175291 Isocitrate dehydrogenase [NADP], mitochondrial Proteins 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000012165 high-throughput sequencing Methods 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 206010069754 Acquired gene mutation Diseases 0.000 description 1
- 208000005243 Chondrosarcoma Diseases 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 230000007067 DNA methylation Effects 0.000 description 1
- 101000960235 Dictyostelium discoideum Isocitrate dehydrogenase [NADP] cytoplasmic Proteins 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101000599886 Homo sapiens Isocitrate dehydrogenase [NADP], mitochondrial Proteins 0.000 description 1
- 108010075869 Isocitrate Dehydrogenase Proteins 0.000 description 1
- 102000012011 Isocitrate Dehydrogenase Human genes 0.000 description 1
- 102100037845 Isocitrate dehydrogenase [NADP], mitochondrial Human genes 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000006957 competitive inhibition Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000003831 deregulation Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000001973 epigenetic effect Effects 0.000 description 1
- 230000006718 epigenetic regulation Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- ODBLHEXUDAPZAU-UHFFFAOYSA-N isocitric acid Chemical compound OC(=O)C(O)C(C(O)=O)CC(O)=O ODBLHEXUDAPZAU-UHFFFAOYSA-N 0.000 description 1
- 101150044508 key gene Proteins 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- JMANVNJQNLATNU-UHFFFAOYSA-N oxalonitrile Chemical compound N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 238000005895 oxidative decarboxylation reaction Methods 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000037439 somatic mutation Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q2600/156—Polymorphic or mutational 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 belongs to the technical field of gene detection, and in particular relates to quantitative detectionIDH1Probes, kits and drop-off ddPCR methods for gene mutation. The invention is based onIDH1Designing two probes with different fluorescent signals in a hotspot mutation region of a gene No.4 exon, wherein a Drop-off probe is positioned on the hotspot mutation region, and the probes are complementary with a wild type sequence and are not complementary with mutation sites; the Reference probe is located outside the mutation hot spot region and is complementary to both the mutant and wild type sequences. As long as the Drop-off probe coverage area is mutated, the probe cannot be combined with a template sequence, the Reference probe can still be combined with the probe and amplified, and finally the mutation type can be distinguished by the fluorescence signal intensity of the liquid Drop measured by a detection instrument and the position in two-dimensional coordinates,Wild type and negative, realizationIDH1Detection of gene mutation.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a probe, a kit and a drop-off ddPCR method for quantitatively detecting IDH1 gene mutation.
Background
The IDH1 gene is located on chromosome 2 of human genome, is used as key gene involved in cell metabolism, and its coded protein isocitrate dehydrogenase 1 is cytosolic enzyme, mainly consists of 414 amino acids, and can combine two NADP + The molecule and 154 water molecules, and contains C-terminal tripeptide, alanine-lysine-leucine, are type 1 peroxisome targeting sequences, which can target proteins to peroxisomes. The protein is mainly present in cytoplasm, peroxisome and endoplasmic reticulum, and can catalyze oxidative decarboxylation of isocitrate to alpha-ketoglutarate (alpha-KG). NADP consumption by catalytic processes + NADPH is produced, and the catalytic products alpha-KG and NADPH can relieve oxidative damage of cells caused by oxidative stress and play an important role in the process of cell detoxification. In addition to the above functions, IDH1 catalyzes the β -oxidation of unsaturated fatty acids in the peroxisome of hepatocytes.
IDH1 is the most frequent gene for mutation of metabolic genes (metabic genes) in human cancers, and mutant IDH1 interferes with normal metabolism and epigenetic regulation of cells, and is ubiquitous in many myeloid malignancies, and studies indicate that IDH1 (R132C and R132H) or IDH2 (R172 and R140) somatic mutations are detected in about 20% of patients with Acute Myeloid Leukemia (AML). While normal IDH1 protein plays a key role in the food nutrition metabolism process, mutant IDH1 protein in AML patients additionally converts alpha-KG produced by the catalysis of IDH1 into metabolite 2-hydroxyglutarate (2-HG) during the metabolism process, and the process consumes NADPH to generate NADP + .2-HG is an oncogenic molecule, and the latter can inhibit the former target due to competitive inhibition caused by the structural similarity of alpha-KG and 2-HG, thus, the alpha-KG is used as a base in AML patientsThe signaling pathways of the agent are often disrupted, resulting in epigenetic deregulation of histone and DNA methylation, chromatin remodeling, blocking of cell differentiation and other transformation effects, leading to the development of tumors. In addition, besides AML, research shows that IDH1 mutation is closely related to diseases such as glioma, cartilage tumor, chondrosarcoma, glioma and the like, and IDH genes are already listed in NCCN guidelines and Chinese glioma guidelines, which clearly indicate that IDH1/2 is a key index of molecular typing of glioma, and has important significance for individual treatment and clinical prognosis judgment of glioma.
There are various detection methods for IDH1 gene mutation, mainly Sanger sequencing, second generation sequencing, real-time fluorescent quantitative PCR, traditional ddPCR (microdroplet digital PCR, droplet Digital PCR) and the like. Among them, sanger sequencing is the most classical method for detecting IDH1 gene mutation and is also the gold standard for detecting gene mutation. The direct sequencing method based on the dideoxy sequencing principle can most intuitively show the change of the gene sequence in the form of a base peak diagram, has comprehensive detection types and is also the earliest mutation detection means. The second generation sequencing has the advantages of high flux, high sensitivity and the like, but the experimental operation is difficult and the cost is high. The real-time fluorescent quantitative PCR is a detection method for judging whether mutation exists or not by using a Ct value based on the PCR, and has the advantages of strong specificity, high sensitivity, accurate quantification, simple and convenient operation, full-closed reaction and the like, but absolute quantification cannot be performed and the risk of false positive possibly exists. The traditional ddPCR method has high sensitivity, can directly reflect the mutation load level of IDH1, but can only detect one known mutation by a pair of probes, can not detect multiple mutations on the same hot spot at the same time, and can possibly generate omission.
Disclosure of Invention
The invention aims at a probe, a kit and a drop-off ddPCR method for quantitatively detecting IDH1 gene mutation, and the primer and the probe can realize detection of a plurality of known mutation sites on the IDH1 gene, and have high sensitivity and simple test operation.
The invention provides a probe for detecting IDH1 gene mutation, which comprises a Drop-off probe and a Reference probe, wherein the nucleotide sequence of the Drop-off probe is shown as SEQ ID NO.1, and the nucleotide sequence of the Reference probe is shown as SEQ ID NO. 2; the 5 'end of the Drop-off probe and the Reference probe is marked with a fluorescent group, and the 3' end is marked with a fluorescence quenching group; the Drop-off probe and the Reference probe bear different fluorophores.
Preferably, the 5 'end of the Drop-off probe is marked with a fluorescent group VIC, and the 3' end is marked with a fluorescence quenching group BHQ1; the Reference probe is marked with a fluorescent group FAM at the 5 'end and a fluorescence quenching group BHQ1 at the 3' end.
The invention also provides a primer for detecting IDH1 gene mutation, the nucleotide sequence of the forward primer of the primer is shown as SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 4.
The invention also provides a drop-off ddPCR kit for quantitatively detecting IDH1 gene mutation, which comprises the probe and the primer for detecting IDH1 gene mutation.
Preferably, the primer for detecting IDH1 gene mutation comprises the primer according to the technical scheme.
Preferably, the kit further comprises a dPCR enzyme and a buffer.
Preferably, the kit further comprises an IDH1 mutant plasmid and an IDH1 wild-type plasmid.
The invention also provides application of the probe, the primer or the kit in preparation of products for detecting IDH1 gene mutation.
Preferably, the IDH1 gene mutation comprises one or more of R132C, R132G, R132H, R L and R132S.
The invention also provides a drop-off ddPCR method for quantitatively detecting IDH1 gene mutation, which comprises the following steps:
mixing genomic DNA of a sample to be detected, a primer, a probe, dPCR enzyme and a buffer solution to obtain ddPCR reaction mixed solution;
mixing the ddPCR reaction mixed solution with an oil phase, and preparing the obtained mixture by a droplet preparation instrument to generate micro-reaction droplets;
introducing the micro-reaction liquid drops into a chip, performing PCR amplification reaction, collecting signals of the micro-droplets in the chip after the PCR amplification reaction, and calculating to obtain the mutation frequency of the IDH1 gene in the sample to be detected;
the probe comprises the probe according to the technical scheme.
The beneficial effects are that:
the invention provides a probe for detecting IDH1 gene mutation, which comprises a Drop-off probe and a Reference probe, wherein the nucleotide sequence of the Drop-off probe is shown as SEQ ID NO.1, and the nucleotide sequence of the Reference probe is shown as SEQ ID NO. 2; the 5 'end of the Drop-off probe and the Reference probe is marked with a fluorescent group, and the 3' end is marked with a fluorescence quenching group; the Drop-off probe and the Reference probe bear different fluorophores. According to the invention, two probes with different fluorescent signals are designed according to the hot spot mutation region of the No.4 exon of the IDH1 gene, wherein the Drop-off probes are positioned on the hot spot mutation region, and the probes are complementary with a wild type sequence and are not complementary with mutation sites; the Reference probe is located outside the mutation hot spot region and is complementary to both the mutant and wild type sequences. As long as the Drop-off probe coverage area is mutated, the probe cannot be combined with a template sequence, and the Reference probe can still be combined and amplified at the moment, and finally, the mutation type, the wild type and the negative type can be distinguished according to the fluorescence signal intensity of the liquid Drop measured by a detection instrument and the position of the liquid Drop in two-dimensional coordinates; the wild type carries two fluorescent signals, namely a Drop-off probe and a Reference probe; mutant forms carry only the Reference probe fluorescent signal; negative with no fluorescent signal.
On the basis, the detection of multiple mutations of the mutation hot spot of the 4 th exon of the IDH1 gene can be realized by combining primer amplification, and compared with Sanger sequencing, the detection method has high sensitivity and is simpler than the second generation sequencing experiment operation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a 2D scattergram of IDH1 gene mutation detection in example 1;
FIG. 2 is a 2D scatter plot of the IDH1 mutant type plasmid assay of example 2;
FIG. 3 is a plot of the detected 2D scatter plot after dilution of the wild-type plasmid to 1% in example 2;
FIG. 4 is a plot of the detected 2D scatter plot after dilution of the wild-type plasmid to 0.5% in example 2;
FIG. 5 is a plot of the detected 2D scatter plot after dilution of the wild-type plasmid to 0.2% in example 2;
FIG. 6 is a plot of the detected 2D scatter plot after dilution of the wild-type plasmid to 0.1% in example 2;
FIG. 7 is a plot of the detected 2D scatter plot after dilution of the wild-type plasmid to 0.05% in example 2;
FIG. 8 is a 2D scatter plot of the negative samples of example 2;
FIG. 9 is a linear relationship between the theoretical mutation frequency and the actual mutation frequency of IDH1 in example 2.
Detailed Description
The invention provides a probe which comprises a Drop-off probe and a Reference probe, wherein the nucleotide sequence of the Drop-off probe is shown as SEQ ID NO.1, and the nucleotide sequence of the Reference probe is shown as SEQ ID NO. 2; the 5 'end of the Drop-off probe and the Reference probe is marked with a fluorescent group, and the 3' end is marked with a fluorescence quenching group; the Drop-off probe and the Reference probe bear different fluorophores.
In the invention, the nucleotide sequence of the Drop-off probe is shown as SEQ ID NO.1, specifically 5'-TGGGTAAAACCTATCATCATAGGTC-3', the 5 'end of the Drop-off probe is preferably marked with a fluorescent group VIC, the 3' end of the Drop-off probe is preferably marked with a fluorescent quenching group BHQ1, and the sequence after marking the fluorescent group is as follows: 5'-VIC-TGGGTAAAACCTATCATCATAGGTC-BHQ1-3'. The nucleotide sequence of the Reference probe is shown as SEQ ID NO.2, specifically 5'-ATGGGGATCAAGTAAGTCATGTTGG-3', the 5 'end of the Reference probe is preferably marked with a fluorescent group FAM, the 3' end of the Reference probe is marked with a fluorescence quenching group BHQ1, and the marked sequence is as follows: 5'-FAM-ATGGGGATCAAGTAAGTCATGTTGG-BHQ1-3'.
According to the invention, two probes are designed aiming at the mutation hot spot region of the No.4 exon of the IDH1 gene, one probe is positioned outside the mutation hot spot region, when the mutation hot spot region generates base mutation, deletion and insertion, the Drop-off probe positioned in the mutation hot spot region cannot be tightly combined with a template, and the Reference probe can still be combined and amplified with the template at the moment, so that the detection of multiple mutation of the mutation hot spot of the No.4 exon of the IDH1 gene, including unknown mutation sites, is realized.
The invention also provides a primer for detecting IDH1 gene mutation, the nucleotide sequence of the forward primer of the primer is shown as SEQ ID NO.3, and the primer specifically comprises the following components: 5'-GCCATTATCTGCAAAAATATCCCCC-3' the nucleotide sequence of the reverse primer is shown as SEQ ID NO.4, and specifically comprises the following steps: 5'-TACAAGTTGGAAATTTCTGGGCCAT-3'.
The invention also provides a drop-off ddPCR kit for quantitatively detecting IDH1 gene mutation, which comprises the probe and the primer for detecting IDH1 gene mutation. The primer for detecting IDH1 gene mutation preferably comprises the primer according to the technical scheme. The kit of the present invention preferably further comprises a dPCR enzyme and a buffer, the source and brand of which are not particularly limited, and reagents conventionally used in the art for drop-off ddPCR detection may be employed. The kit of the invention preferably further comprises an IDH1 mutant plasmid and an IDH1 wild type plasmid; the IDH1 mutant plasmid and the IDH1 wild type plasmid can be used for positive control and negative control for detecting IDH1 gene mutation respectively.
The invention also provides application of the probe, the primer or the kit in preparation of products for detecting IDH1 gene mutation. The known mutation site in the IDH1 gene mutation of the present invention preferably includes one or more of R132C, R132G, R132H, R L and R132S, more preferably a plurality of R132C, R132G, R132H, R L and R132S, and still more preferably R132C, R132G, R132H, R L and R132S.
The invention also provides a drop-off ddPCR method for quantitatively detecting IDH1 gene mutation, which comprises the following steps:
mixing genomic DNA of a sample to be detected, a primer, a probe, dPCR enzyme and a buffer solution to obtain ddPCR reaction mixed solution;
mixing the ddPCR reaction mixed solution with an oil phase, and preparing the obtained mixture by a droplet preparation instrument to generate micro-reaction droplets;
introducing the micro-reaction liquid drops into a chip, performing PCR amplification reaction, collecting signals of the micro-droplets in the chip after the PCR amplification reaction, and calculating to obtain the mutation frequency of the IDH1 gene in the sample to be detected;
the probe comprises the probe according to the technical scheme.
The invention mixes the genome DNA of the sample to be tested, the primer, the probe, the dPCR enzyme and the buffer solution to obtain the ddPCR reaction mixed solution. The specific step of extracting the genomic DNA of the sample to be detected is not particularly limited, and the genomic DNA can be extracted by adopting a conventional extraction method in the field, such as extraction by adopting a kit. The genome DNA, the primer, the probe, the dPCR enzyme and the buffer solution of the sample to be tested form the amplification system of the drop-off ddPCR, and the volume of each component is preferably as follows according to 30 mu L: 1-15. Mu.L of genomic DNA of a sample to be tested, 7.5. Mu.L of 4 XMaxMix, 3.3. Mu.L of upstream primer, 3.3. Mu. L, drop-off probe 0.45. Mu. L, reference probe 0.45. Mu.L of downstream primer and the balance of water. The primer of the invention preferably comprises the primers shown in SEQ ID NO.3 and SEQ ID NO. 4.
After the ddPCR reaction mixture is obtained, the ddPCR reaction mixture is mixed with an oil phase. The volume ratio of the ddPCR reaction mixed solution to the oil phase is preferably 3:16.
after the mixing, the micro-reaction liquid drops are prepared from the obtained mixture by a micro-drop preparation instrument. The steps and processes for preparing the micro-reactive droplets are not particularly limited, and conventional steps and processes in the art may be employed.
After the micro-reaction liquid drops are obtained, the micro-reaction liquid drops are led into a chip for PCR amplification reaction, and the micro-droplets in the chip after the PCR amplification reaction are subjected to signal collection, so that the IDH1 gene mutation frequency in the sample to be detected is calculated, wherein the calculation formula of the gene mutation frequency is FAM copy number/(FAM copy number+VIC copy number). The PCR amplification procedure of the present invention preferably comprises: hot start at 95 ℃ for 10min; denaturation at 94℃for 30s, annealing at 60℃for 1min, total 40 cycles, and instrument cooling at 12℃for 5min.
The drop-off ddPCR method of the present invention preferably includes a drop-off ddPCR method for diagnostic or non-diagnostic purposes.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
A drop-off ddPCR kit and a method for quantitatively detecting IDH1 gene mutation comprise the following steps:
1 example of tissue DNA for detecting IDH 1R 132H gene mutation by high throughput sequencing (NGS) is selected, and a using method of the kit is introduced.
The kit combines a liquid drop type digital PCR technology with a Taqman fluorescent probe method, and detects 5 mutations on the No.4 exon of the IDH1 gene, wherein specific mutation sites comprise: R132C, R132G, R H, R132L, R132S.
The kit comprises 2 reagents, wherein the reagent 1 mainly comprises the following components: enzyme, buffer, and enzyme and buffer were purchased from Beijing New Biotechnology Co., ltd., product number 23010, 380. Mu.L/tube; the reagent 2 mainly comprises the following components: a primer and a probe; detecting the sample type: FFPE organization.
The primer is as follows: IDH1 forward primer: 5'-GCCATTATCTGCAAAAATATCCCCC-3', IDH1 reverse primer: 5'-TACAAGTTGGAAATTTCTGGGCCAT-3';
the probe is as follows: drop-off probe: 5'-VIC-TGGGTAAAACCTATCATCATAGGTC-BHQ1-3', reference probe: 5'-FAM-ATGGGGATCAAGTAAGTCATGTTGG-BHQ1-3'.
The specific experimental method comprises the following steps:
1. extraction of tissue DNA: DNA was extracted according to the tissue DNA extraction kit procedure, and the extracted DNA was quantified using Qubit, QIAxcel, to detect fragment size.
2. Digital PCR detection
The PCR reaction system was prepared as shown in Table 1 using the DNA template extracted in step 1, wherein 4 XMaxMix was purchased from Biotechnology Inc. of Beijing as a finished product reagent, and the product number was 23010.
TABLE 1 PCR reaction System
The prepared PCR reaction system was mixed with 10. 10 s, and the mixture was subjected to instantaneous centrifugation.
3. Micro-droplet preparation
The reaction system was prepared by adding 160. Mu.L of the droplet-forming oil and 30. Mu.L of the above-described reaction system, in accordance with the instructions of the micro-droplet-forming kit (kit model 10001 available from Biotechnology Co., ltd. In Beijing).
4. PCR amplification
The resulting 8 rows containing microdroplets were capped and placed on a PCR apparatus for amplification, and the PCR amplification procedure is shown in Table 2.
TABLE 2 PCR amplification procedure
5. Result detection
And (3) placing the amplified chip on a biological reader for detection, and analyzing the data to obtain a final 2D scatter diagram (figure 1) and FAM and VIC related data.
The result shows that: in 30ng of DNA, the FAM copy number was 2469 copies, the VIC copy number was 10264.6 copies, and the mutation frequency was 19.39% as calculated by the formula of mutation frequency=FAM/(FAM+VIC), and positive. It can be seen that the results of the detection using the method of the present invention are consistent with those obtained using high throughput sequencing.
Example 2
The sensitivity test of the kit comprises the following steps:
sensitivity tests were performed using IDH 1-containing mutant plasmid DNA and wild-type plasmid DNA as templates.
Plasmid samples are self-synthesized, and the plasmid construction method comprises the following steps: plasmid vector was prepared by cutting vector pUC57 using double cleavage (Nde I/Sph I); screening NGS to detect a sample containing IDH1 hot spot mutation, designing a primer according to the sequence of a mutation region, and designing a forward primer: 5'-TGAGATGGACGCCTATTTGT-3' (SEQ ID NO. 5), reverse primer: 5'-GTGATGCCACCAACGACCAA-3' (SEQ ID NO. 6); amplifying and enriching the target gene fragment by using a pcr method, and detecting the accuracy of the size of the target gene fragment by using agarose gel electrophoresis; respectively recovering plasmid vector DNA and target gene DNA fragments after electrophoresis, linking the vector and the target gene by using T4 DNA ligase, adding a connection product into competent cells DH5 alpha, standing on ice for 30min, performing heat shock conversion at 42 ℃, adding LB culture medium, shaking for 45-60min by a shaking table at 200rpm and 37 ℃, coating onto a screening plate, culturing overnight at 37 ℃, picking up 5 single colonies, performing enzyme digestion identification, and carrying out first generation sequencing for further verification after identification.
The quantitative result of the IDH1 mutant plasmid is 1495.1 copies, and then the wild type plasmid is used to dilute the IDH1 mutant plasmid to 1%, 0.5%, 0.2%, 0.1%, 0.05%, NC (healthy human blood leukocyte DNA), each sample is repeated 3 times, each group of template DNA is detected according to the detection method in example 1, the IDH1 mutation frequency result is shown in table 3, the 2D scatter diagram of each template is shown in fig. 2-8, and the linear relation between the IDH1 theoretical mutation frequency and the actually detected mutation frequency is shown in fig. 9.
TABLE 3 detection results of template DNA of each group
As shown in FIG. 9, the theoretical mutation frequency has a good linear relationship with the actual mutation frequency, R 2 The kit cannot detect the mutation at a theoretical mutation frequency of 0.05%, but the lowest detection frequency of the kit is 0.1%.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. The probe for detecting IDH1 gene mutation is characterized by comprising a Drop-off probe and a Reference probe, wherein the nucleotide sequence of the Drop-off probe is shown as SEQ ID NO.1, and the nucleotide sequence of the Reference probe is shown as SEQ ID NO. 2; the 5 'end of the Drop-off probe and the Reference probe is marked with a fluorescent group, and the 3' end is marked with a fluorescence quenching group; the Drop-off probe and the Reference probe bear different fluorophores.
2. The probe of claim 1, wherein the Drop-off probe is labeled with a fluorescent group VIC at the 5 'end and a fluorescence quenching group BHQ1 at the 3' end; the Reference probe is marked with a fluorescent group FAM at the 5 'end and a fluorescence quenching group BHQ1 at the 3' end.
3. A primer for detecting IDH1 gene mutation is characterized in that the nucleotide sequence of a forward primer of the primer is shown as SEQ ID NO.3, and the nucleotide sequence of a reverse primer is shown as SEQ ID NO. 4.
4. A drop-off ddPCR kit for quantitatively detecting IDH1 gene mutation, wherein the kit comprises the probe of claim 1 or 2 and a primer for detecting IDH1 gene mutation.
5. The kit according to claim 4, wherein the primer for detecting mutation of IDH1 gene is the primer according to claim 3.
6. The kit of claim 4 or 5, further comprising a dPCR enzyme and a buffer.
7. The kit of claim 4 or 5, further comprising an IDH1 mutant plasmid and an IDH1 wild-type plasmid.
8. Use of the probe of claim 1 or 2, the primer of claim 3 or the kit of any one of claims 4 to 7 for the preparation of a product for detecting IDH1 gene mutation.
9. The use of claim 8, wherein the IDH1 gene mutation comprises one or more of R132C, R132G, R132H, R L and R132S.
10. A drop-off ddPCR method for quantitatively detecting IDH1 gene mutation is characterized by comprising the following steps:
mixing genomic DNA of a sample to be detected, a primer, a probe, dPCR enzyme and a buffer solution to obtain ddPCR reaction mixed solution;
mixing the ddPCR reaction mixed solution with an oil phase, and preparing the obtained mixture by a droplet preparation instrument to generate micro-reaction droplets;
introducing the micro-reaction liquid drops into a chip, performing PCR amplification reaction, collecting signals of the micro-droplets in the chip after the PCR amplification reaction, and calculating to obtain the mutation frequency of the IDH1 gene in the sample to be detected;
the probe comprises the probe of claim 1 or 2.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311722602.3A CN117402975A (en) | 2023-12-15 | 2023-12-15 | Probe, kit and drop-off ddPCR method for quantitatively detecting IDH1 gene mutation |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311722602.3A CN117402975A (en) | 2023-12-15 | 2023-12-15 | Probe, kit and drop-off ddPCR method for quantitatively detecting IDH1 gene mutation |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117402975A true CN117402975A (en) | 2024-01-16 |
Family
ID=89487477
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311722602.3A Pending CN117402975A (en) | 2023-12-15 | 2023-12-15 | Probe, kit and drop-off ddPCR method for quantitatively detecting IDH1 gene mutation |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117402975A (en) |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108315432A (en) * | 2018-05-04 | 2018-07-24 | 良培基因生物科技(武汉)有限公司 | DdPCR technologies detect primer, kit and the detection method of IDH1 R132C genetic mutations |
CN108342461A (en) * | 2018-05-04 | 2018-07-31 | 良培基因生物科技(武汉)有限公司 | DdPCR technologies detect primer, kit and the detection method of IDH1 genetic mutations |
WO2019175323A1 (en) * | 2018-03-14 | 2019-09-19 | Universite De Versailles Saint-Quentin-En-Yvelines | Method for identifying one or more mutations in a hotspot mutation sequence |
CN112239787A (en) * | 2019-07-19 | 2021-01-19 | 河南远止生物技术有限公司 | Primer, probe, kit and device for detecting human IDH1 gene mutation |
CN113186283A (en) * | 2021-04-30 | 2021-07-30 | 镇江市第一人民医院 | Drop-off ddPCR method and kit for quantitatively detecting U2AF1 gene mutation |
CN113249475A (en) * | 2021-04-30 | 2021-08-13 | 镇江市第一人民医院 | Drop-off ddPCR method and kit for quantitatively detecting NPM1 gene mutation |
-
2023
- 2023-12-15 CN CN202311722602.3A patent/CN117402975A/en active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019175323A1 (en) * | 2018-03-14 | 2019-09-19 | Universite De Versailles Saint-Quentin-En-Yvelines | Method for identifying one or more mutations in a hotspot mutation sequence |
CN108315432A (en) * | 2018-05-04 | 2018-07-24 | 良培基因生物科技(武汉)有限公司 | DdPCR technologies detect primer, kit and the detection method of IDH1 R132C genetic mutations |
CN108342461A (en) * | 2018-05-04 | 2018-07-31 | 良培基因生物科技(武汉)有限公司 | DdPCR technologies detect primer, kit and the detection method of IDH1 genetic mutations |
CN112239787A (en) * | 2019-07-19 | 2021-01-19 | 河南远止生物技术有限公司 | Primer, probe, kit and device for detecting human IDH1 gene mutation |
CN113186283A (en) * | 2021-04-30 | 2021-07-30 | 镇江市第一人民医院 | Drop-off ddPCR method and kit for quantitatively detecting U2AF1 gene mutation |
CN113249475A (en) * | 2021-04-30 | 2021-08-13 | 镇江市第一人民医院 | Drop-off ddPCR method and kit for quantitatively detecting NPM1 gene mutation |
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 | |
CN110878343B (en) | Cpf1 kit for rapidly detecting genetic deafness pathogenic gene SLC26A4 mutation and detection method thereof | |
CN110964814B (en) | Primers, compositions and methods for nucleic acid sequence variation detection | |
CN108251510A (en) | A kind of kit, detection method and its application of folic acid metabolism ability Genotyping | |
CN111235272A (en) | Composition for one-time detection of lung cancer multiple gene mutation and application thereof | |
CN109913559B (en) | RYR2 gene as molecular marker influencing sheep feed conversion rate and application thereof | |
CN111647649B (en) | Method for assisted selection of cattle growth traits based on CCDC39 gene CNV detection | |
CN113136429A (en) | Detection kit and detection method for IDH1 or IDH2 gene mutation | |
CN111334580A (en) | PIK3CA gene mutation detection kit | |
CN108342461A (en) | DdPCR technologies detect primer, kit and the detection method of IDH1 genetic mutations | |
CN114410844A (en) | Method for determining RCR negative and positive in CAR-T cell product by detecting GALV and ALB copy number through multiple qPCR (quantitative polymerase chain reaction) | |
CN115029444A (en) | Molecular marker related to sheep growth traits and application thereof | |
CN113186283B (en) | Drop-off ddPCR method and kit for quantitatively detecting U2AF1 gene mutation | |
CN113502335A (en) | Molecular marker related to sheep growth traits and application thereof | |
CN110607381B (en) | Mycobacterium tuberculosis detection kit and method | |
CN112921101A (en) | Molecular marker related to sheep remaining feed intake and application thereof | |
CN109439704B (en) | Method and kit for detecting leukemia related gene variation | |
CN111500720A (en) | PIK3CA gene mutation detection method and kit thereof | |
CN108796077B (en) | Primer pair group and kit for detecting cytosine deaminase and related molecular gene modification difference in cfDNA | |
CN117402975A (en) | Probe, kit and drop-off ddPCR method for quantitatively detecting IDH1 gene mutation | |
CN115044682A (en) | Molecular marker related to growth traits of Hu sheep, detection method and application thereof | |
CN109136367A (en) | The method for improving the diagnosis efficiency of BRAF gene V600E mutation | |
CN113106156A (en) | IDH1R132H gene mutation real-time fluorescence quantitative PCR detection reagent and method | |
CN112029833A (en) | Rapid identification method of CTNNB1 gene mutation for tumor organoid culture condition selection | |
CN110616261A (en) | Kit and method for detecting EGFR gene T790M mutation |
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 |