CN102367478B - ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and detection method - Google Patents
ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and detection method Download PDFInfo
- Publication number
- CN102367478B CN102367478B CN 201110327916 CN201110327916A CN102367478B CN 102367478 B CN102367478 B CN 102367478B CN 201110327916 CN201110327916 CN 201110327916 CN 201110327916 A CN201110327916 A CN 201110327916A CN 102367478 B CN102367478 B CN 102367478B
- Authority
- CN
- China
- Prior art keywords
- seq
- primer
- arms
- kras
- qpcr
- 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.)
- Expired - Fee Related
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 49
- 230000035772 mutation Effects 0.000 title claims abstract description 24
- 206010064571 Gene mutation Diseases 0.000 title claims abstract description 14
- 102100030708 GTPase KRas Human genes 0.000 title claims description 33
- 101000584612 Homo sapiens GTPase KRas Proteins 0.000 title claims description 33
- 238000003753 real-time PCR Methods 0.000 title description 3
- 108700028369 Alleles Proteins 0.000 title 1
- 239000000523 sample Substances 0.000 claims abstract description 78
- 101150105104 Kras gene Proteins 0.000 claims abstract description 54
- 238000011529 RT qPCR Methods 0.000 claims abstract description 37
- 238000006243 chemical reaction Methods 0.000 claims abstract description 32
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 19
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 19
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 19
- 102000004190 Enzymes Human genes 0.000 claims abstract description 13
- 108090000790 Enzymes Proteins 0.000 claims abstract description 13
- 239000013641 positive control Substances 0.000 claims abstract description 8
- 108020004705 Codon Proteins 0.000 claims description 38
- 108090000623 proteins and genes Proteins 0.000 claims description 20
- 238000011144 upstream manufacturing Methods 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 7
- 238000013016 damping Methods 0.000 claims description 5
- 239000012530 fluid Substances 0.000 claims description 5
- 206010028980 Neoplasm Diseases 0.000 abstract description 21
- 238000012163 sequencing technique Methods 0.000 abstract description 9
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 201000011510 cancer Diseases 0.000 abstract description 7
- 230000008901 benefit Effects 0.000 abstract description 6
- 210000002966 serum Anatomy 0.000 abstract description 6
- 210000001124 body fluid Anatomy 0.000 abstract description 3
- 239000010839 body fluid Substances 0.000 abstract description 3
- 239000012634 fragment Substances 0.000 abstract description 3
- 230000000903 blocking effect Effects 0.000 abstract 1
- 230000001413 cellular effect Effects 0.000 abstract 1
- 230000008859 change Effects 0.000 description 46
- 108020004414 DNA Proteins 0.000 description 41
- 230000003321 amplification Effects 0.000 description 30
- 238000003199 nucleic acid amplification method Methods 0.000 description 30
- 230000037429 base substitution Effects 0.000 description 12
- 238000000034 method Methods 0.000 description 12
- 238000013461 design Methods 0.000 description 11
- 206010009944 Colon cancer Diseases 0.000 description 9
- 210000002381 plasma Anatomy 0.000 description 9
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 8
- 239000002585 base Substances 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- 238000010586 diagram Methods 0.000 description 8
- 230000004087 circulation Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 239000003471 mutagenic agent Substances 0.000 description 6
- 238000005457 optimization Methods 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 229960005395 cetuximab Drugs 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 208000005016 Intestinal Neoplasms Diseases 0.000 description 4
- 238000000137 annealing Methods 0.000 description 4
- 201000002313 intestinal cancer Diseases 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000005551 L01XE03 - Erlotinib Substances 0.000 description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- AAKJLRGGTJKAMG-UHFFFAOYSA-N erlotinib Chemical compound C=12C=C(OCCOC)C(OCCOC)=CC2=NC=NC=1NC1=CC=CC(C#C)=C1 AAKJLRGGTJKAMG-UHFFFAOYSA-N 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 201000005202 lung cancer Diseases 0.000 description 3
- 208000020816 lung neoplasm Diseases 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 125000006853 reporter group Chemical group 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 230000010558 Gene Alterations Effects 0.000 description 2
- 101150040459 RAS gene Proteins 0.000 description 2
- 101150112897 TS gene Proteins 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 229940082789 erbitux Drugs 0.000 description 2
- -1 fresh frozen tissue Substances 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 230000003211 malignant effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- JMANVNJQNLATNU-UHFFFAOYSA-N oxalonitrile Chemical compound N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 description 2
- 229960001972 panitumumab Drugs 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- 239000004575 stone Substances 0.000 description 2
- 229940120982 tarceva Drugs 0.000 description 2
- 239000001993 wax Substances 0.000 description 2
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 206010052358 Colorectal cancer metastatic Diseases 0.000 description 1
- 241001301450 Crocidium multicaule Species 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 102000013446 GTP Phosphohydrolases Human genes 0.000 description 1
- 102100029974 GTPase HRas Human genes 0.000 description 1
- 102100039788 GTPase NRas Human genes 0.000 description 1
- 108091006109 GTPases Proteins 0.000 description 1
- 101000584633 Homo sapiens GTPase HRas Proteins 0.000 description 1
- 101000744505 Homo sapiens GTPase NRas Proteins 0.000 description 1
- 101150117869 Hras gene Proteins 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 108700020978 Proto-Oncogene Proteins 0.000 description 1
- 102000052575 Proto-Oncogene Human genes 0.000 description 1
- 241000713810 Rat sarcoma virus Species 0.000 description 1
- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- 206010038019 Rectal adenocarcinoma Diseases 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000001531 bladder carcinoma Diseases 0.000 description 1
- 238000004159 blood analysis Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 229940121647 egfr inhibitor Drugs 0.000 description 1
- 229960001433 erlotinib Drugs 0.000 description 1
- 208000021045 exocrine pancreatic carcinoma Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 150000002243 furanoses Chemical class 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 231100001261 hazardous Toxicity 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000011337 individualized treatment Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 230000001473 noxious effect Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 210000004976 peripheral blood cell Anatomy 0.000 description 1
- 239000002831 pharmacologic agent Substances 0.000 description 1
- 238000012175 pyrosequencing Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 108700042226 ras Genes Proteins 0.000 description 1
- 102000016914 ras Proteins Human genes 0.000 description 1
- 206010038038 rectal cancer Diseases 0.000 description 1
- 201000001281 rectum adenocarcinoma Diseases 0.000 description 1
- 201000001275 rectum cancer Diseases 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 208000010570 urinary bladder carcinoma Diseases 0.000 description 1
- 230000004304 visual acuity Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及涉及分子生物学领域，旨在提供一种用于KRAS基因突变分型的ARMS-qPCR检测试剂盒及检测方法。该试剂盒包括qPCR混合反应液、锁核酸阻滞探针、参照引物、ARMS引物和阳性对照样品；其中qPCR混合反应液包括：PCR缓冲液、dNTPs、MgCl2、Goldstarbest Taq酶、PCR通用下游引物和通用TaqMan探针。本发明的试剂盒能快速、准确、高敏感度的检测各种癌组织中KRAS基因特定位点突变。灵敏度高，可以检测各种组织来源的基因组DNA，不仅来源于细胞体系，尤其是采用非细胞体系如血清、血浆或其他体液来源的游离DNA片段。与直接测序等突变检测技术比较，本发明用于检测KRAS基因突变具有特异性强、敏感性高、操作简单快速、高通量、安全、结果判读明确客观等优势。
The invention relates to the field of molecular biology and aims to provide an ARMS-qPCR detection kit and detection method for KRAS gene mutation typing. The kit includes qPCR mixed reaction solution, locked nucleic acid blocking probe, reference primers, ARMS primers and positive control samples; the qPCR mixed reaction solution includes: PCR buffer, dNTPs, MgCl 2 , Goldstarbest Taq enzyme, PCR universal downstream primers and universal TaqMan probes. The kit of the invention can quickly, accurately and highly sensitively detect mutations at specific sites of the KRAS gene in various cancer tissues. With high sensitivity, it can detect genomic DNA from various tissue sources, not only from cell systems, especially free DNA fragments from non-cellular systems such as serum, plasma or other body fluids. Compared with mutation detection techniques such as direct sequencing, the invention has the advantages of strong specificity, high sensitivity, simple and fast operation, high throughput, safety, and clear and objective result interpretation for detecting KRAS gene mutation.
Description
Technical field
The present invention relates to biology field, be specifically related to a kind of quick detection kit and detection method thereof that instructs the KRAS gene mutation typing of tumour individualized treatment.
Background technology
The RAS proto-oncogene is the transforming gene that the clone comes out from rat sarcoma virus at first, after in people finders' such as nineteen eighty-two Weinberg transitional cell bladder carcinoma cell line, activatory HRAS gene being arranged, caused the very big concern of people to RAS oncogene role in human tumor generation evolution.The gene that the RAS gene family is relevant with human tumor has three kinds---and HRAS, KRAS and NRAS are positioned at respectively on 11,12 and No. 1 karyomit(e).Wherein, KRAS has the greatest impact to human cancer, and it is just as a kind of molecular switch: when the path that just often can control the regulating cell growth; Take place unusually during as the KRAS transgenation, this gene forever activates, and makes signal conduction disturbance in the cell, causes cell to continue growth, thereby and stops apoptosis generation canceration.
The KRAS transgenation occurs in the early stage of tumour, and the KRAS gene height of primary tumor and metastasis is consistent.It is generally acknowledged that KRAS gene state can not change because of treatment.Sudden change has taken place in the KRAS gene in kinds of tumors, its incidence is about 40% in colorectal cancer, and carcinoma of the pancreas is that lung cancer is about 15% more than 90%.12 and 13 codons (〉=90%) that occur in the 2nd exon are mainly concentrated in the KRAS transgenation, and these sudden changes have destroyed KRAS albumen intrinsic GTPase activity, thereby make KRAS albumen be in the continuous activity state.
The detection KRAS transgenation of rapid sensitive has great clinical meaning: the KRAS gene unconventionality occurs in (1) normal people's blood examination, prompting belongs to the tumour high risk population; (2) if the innocent tumour patient detects the KRAS transgenation, prompting has the possibility that cancerates; (3) a large amount of multicenter 3 clinical trial phases show that target new drug Erbitux (Erbitux, Cetuximab) and handkerchief Buddhist nun monoclonal antibody (Panitumumab) are only applicable to not have the KRAS gene wild-type patient of sudden change, and are invalid to KRAS sudden change patient." the state-run cancer integrated network of the U.S. (NCCN) colorectal cancer clinical practice guideline " (2008 the 3rd edition) spell out: the one, and all metastatic colorectal cancer patients all should detect KRAS gene state; The 2nd, have only KRAS wild-type patient just to advise accepting the treatment of EGFR inhibitor such as Cetuximab (Cetuximab) and handkerchief Buddhist nun monoclonal antibody (panitumumab); Just say as U.S. Georgetown University Marshall: in the treatment of colorectal cancer, from then on colorectal cancer " is divided into two ": the KRAS gene is wild-type or mutant, makes traditional a kind of disease be divided into two kinds of independently diseases.KRAS mutant colorectal cancer patients accounts for 40%, and this class patient can not benefit from anti-EGFR targeted drug treatment, increases untoward reaction danger and medical expense on the contrary on foot; And all the other KRAS wild-type patients of about 60% just probably benefit from this class pharmacological agent." 09 year NCCN nonsmall-cell lung cancer clinical practice guideline " spells out: if sudden change has taken place when the KRAS gene, (Tarceva/ Tarceva/Erlotinib) carries out molecular targeted treatment then not advise patient using Te Luokai.Therefore, whether detection patient KRAS gene suddenlys change and becomes the prerequisite condition that can decision use anti-EGFR targeted drug.
Have high-caliber circulation dissociative DNA in the malignant neoplastic disease human peripheral, this DNA derives from malignant cell.Recently research has found from cancer patients's blood plasma or serum DNA that several TS gene alterations such as KRAS suddenly change, and shows that this is to detect the new way that tumor-related gene changes.Conveniently, the blood plasma Molecular Detection becomes a focus of tumor research rapidly owing to draw materials.But only there is the circulation dissociative DNA of trace in the tumour peripheral blood, and wherein the copy number of mutator gene is lower, so the blood plasma Molecular Detection is higher than organizing to the sensitivity requirement of detection technique.
The method that is used for the KRAS detection in Gene Mutation is a lot, comprises as dna direct order-checking, tetra-sodium order-checking (Pyrosequencing), sex change high performance liquid chromatography (HDPLC), high resolving power solubility curve technology (HRM), restriction small segment length polymorphism analysis method (RFLP) etc.Wherein the dna direct order-checking is the gold standard of sudden change detection.There is following shortcoming in this method: the susceptibility of detection is not high enough: " Lung Cancer " (" lung cancer ") that people such as Katsuhiko once published in August, 2005 goes up report, if the content of mutator gene accounts for 10% when following of genomic dna total amount, then detect existence less than the sudden change sample with direct sequencing; Time-consuming, complicated operating process requires height to operator; The operation of non-stopped pipe relates to the operation behind the pcr amplification, and is therefore easily contaminated, causes that the result's is undesirable; The interpretation subjectivity of sequencing result is strong; Once the sample size of experiment detection is limited, at most can only the 8-24 example.Therefore direct sequencing is difficult to generally carry out clinical.
Have high-caliber circulation dissociative DNA in the malignant neoplastic disease human peripheral, this DNA derives from malignant cell.Recently research has found from cancer patients's blood plasma or serum DNA that several TS gene alterations such as KRAS suddenly change, and shows that this is to detect the new way that tumor-related gene changes.Conveniently, the blood plasma Molecular Detection becomes a focus of tumor research rapidly owing to draw materials.But only there is the circulation dissociative DNA of trace in the tumour peripheral blood, and wherein the copy number of mutator gene is lower, so the blood plasma Molecular Detection is higher than organizing to the sensitivity requirement of detection technique.
Summary of the invention
The technical problem to be solved in the present invention is, overcomes deficiency of the prior art, and a kind of KRAS gene mutation typing ARMS-qPCR detection kit is provided, with this solve in the existing sudden change detection technique, time-consuming, program is loaded down with trivial details and problem such as easy pollution.This test kit can be used for high-throughput, low-cost rapid detection KRAS sudden change, it is highly sensitive, high specificity, applicable to clinical common sample such as fresh frozen tissue, paraffin organization, especially provide the high-sensitivity detection of trace sudden change in atraumatic serum except that pathological tissue or the plasma sample.
Based on above-mentioned purpose, the present invention by the following technical solutions:
A kind of ARMS-qPCR detection kit of the KRAS of being used for gene mutation typing is provided, and this test kit comprises qPCR mixed reaction solution, lock nucleic acid retardance probe, with reference to primer, ARMS primer and positive control sample; Wherein the qPCR mixed reaction solution comprises: PCR damping fluid, dNTPs, MgCl
2, Goldstarbest Taq enzyme, the general downstream primer of PCR and general TaqMan probe;
The final concentration of each component is respectively in the qPCR reaction system: the PCR damping fluid is 1 *; DNTPs is 0.01～1.5mM; MgCl
2Be 0.5～5mM; Goldstarbest Taq enzyme is 0.05U/ μ L; The general downstream primer of PCR is 0.1～1 μ M; General TaqMan probe is 0.1～1 μ M; Lock nucleic acid retardance probe is 0.9 μ M, be that 0.3 μ M, ARMS primer are that 0.3 μ M, positive control sample are 10～300ng/ μ l with reference to primer;
The sequence of the general downstream primer of described PCR is shown in SEQ ID NO A1;
The sequence of described general TaqMan probe is shown in SEQ ID NO A2;
Described lock nucleic acid retardance probe and wild template are mated fully, part base lock nucleination, and its terminal phosphateization, thus suppress KRAS transgenation district wild-type DNA cloning.Its sequence is shown in SEQ ID NO A0;
Described is the upstream primer of can increase simultaneously a KRAS wild-type and a mutated genes group DNA with reference to primer, and its sequence is shown in SEQ ID NO A3;
Described ARMS primer is any one in seven kinds of upstream primers, is used for the corresponding respectively KRAS of detection gene the 12nd codon GAT, GTT, GCT, TGT, AGT and these six kinds of mutants of CGT and No. 13 this a kind of mutation type of codon GAC; The sequence of these seven kinds of upstream primers is shown in SEQ ID NO A4, SEQ ID NO A5, SEQ ID NO A6, SEQ ID NO A7, SEQ ID NO A8, SEQ ID NO A9, SEQ ID NO A10; Article 7, the ARMS primer in the mutating alkali yl coupling of 3 ' terminal bases and mutant to be detected, increases one or two base mispairings in its 3 ' terminal 2-3 reciprocal position simultaneously, to increase its specificity respectively; Be used for six kinds of base substitution mutation (GGT → GAT of specific amplification KRAS gene the 12nd codon, GGT → GTT, GGT → GCT, 12GGT → TGT, the template DNA of GGT → AGT and GGT → CGT) and a kind of base substitution mutation of the 13rd codon (template DNA of codon 13GGC → GAC).
Described positive control sample is the plasmon DNA that has KRAS gene six kinds of mutation types of No. 12 codon and a kind of base substitution mutation type of 13 codons respectively.
Each primer sequence is listed below:
SEQ?ID?NO?A0：5’-TGGAGCTG
GTG
GCGTAGGC-PO4-3’
SEQ?ID?NO?A1：5’-ACCTCTATTGTTGGATCATATTCGTC-3’
SEQ?ID?NO?A2：5’-FAM-GAATTAGCTGTATCGTCAAGGCACTCT-BHQ1-3’
SEQ?ID?NO?A3：5’-CTGAATATAAACTTGTGGTAGTT-3’
SEQ?ID?NO?A4：5’-CTTGTGGTAGTTGGAGCTTA-3’
SEQ?ID?NO?A5：5’-AAACTTGTGGTAGTTGGAGCGGT-3’
SEQ?ID?NO?A6：5’-AACTTGTGGTAGTTGGAGCTGC-3’
SEQ?ID?NO?A7：5’-ATAAACTTGTGGTAGTTGGAGCTA-3’
SEQ?ID?NO?A8：5’-AACTTGTGGTAGTTGGAGCGT-3’
SEQ?ID?NO?A9：5’-ATAAACTTGTGGTAGTTGGAGCCC-3’
SEQ?ID?NO?A10：13Asp-3：5’-GTGGTAGTTGGAGCTGGTAA-3’
Further, the present invention also provides the KRAS gene mutation typing ARMS-qPCR detection method based on the aforementioned agents box, this method is a pair of with reference to primer, TaqMan probe and lock nucleic acid retardance probe at the KRAS gene design earlier, design the ARMS primer respectively at the KRAS gene mutation site, add the qPCR mixed reaction solution in the reaction system, carry out the detection of quantitative fluorescent PCR with reference to primer or ARMS primer, lock nucleic acid retardance probe and template DNA to be checked.Specifically may further comprise the steps:
(1) design and the screening general TaqMan probe, the lock nucleic acid that contain No. the 12nd, KRAS gene and No. 13 codons block probe, with reference to primer and 7 kinds of ARMS primers;
(2) genomic dna that extracts from acellular system or cell system is as template DNA;
(3) carry out the real-time fluorescence quantitative PCR amplification: the sudden change of each sample detects and divides 8 pipes to carry out; Add identical qPCR mixed reaction solution, lock nucleic acid retardance probe and template DNA in every pipe, every pipe adds a kind of with reference in primer or the 7 kinds of ARMS primers respectively, and the ARMS-qPCR that carries out the KRAS gene mutation typing detects;
(4) interpretation as a result:
With reference to the primer pipe amplification curve positive, various ARMS primer Guan Junwu amplification curves or itself and with reference to amplification curve Δ ct＞10 of primer pipe, this sample results is judged as wild-type;
With reference to the primer pipe amplification curve positive, the corresponding ARMS primer pipe amplification curve positive, and itself and with reference to amplification curve Δ ct≤7 of primer pipe, this sample results interpretation is a mutant;
With reference to the primer pipe amplification curve positive, the corresponding ARMS primer pipe amplification curve positive, and itself and with reference to amplification curve Δ ct＞7 of primer pipe, this sample results interpretation is suspicious mutant; All show the amplification curve positive 3 times as duplicate detection, this sample results interpretation is a mutant;
With reference to primer pipe amplification curve feminine gender, point out this sample extraction failure, need to extract again.
Template DNA is selected to extract in the cast of fresh freezing or paraffin-embedded tumor tissues peripheral blood cells, peripheral blood serum or blood plasma, body fluid, cavity in the described step (2).
The condition of carrying out pcr amplification reaction in the described step (3) is: 92～97 ℃ of pre-sex change 10～15min; 92～97 ℃ of sex change 5～15s, 58～64 ℃ of annealing 40s, 40～50 circulations.
With respect to prior art, beneficial effect of the present invention is:
KRAS gene specific locus mutation in the various cancerous tissues of test kit quick, accurate, the high sensitive detection of energy of the present invention.Highly sensitive, can detect various tissue-derived genomic dnas, not only derive from cell system, especially adopt the dissociative DNA fragment in acellular system such as serum, blood plasma or other body fluid source.
Lock nucleic acid (locked nucleic acid, LNA) be a kind of novel few nucleic acid derivative, in the structure 2 one 0 of B-D one ribofuranose, the 4C position forms annular oxygen methylene bridge, sulphur methylene bridge or amine methylene bridge by the shrink effect, the structure of furanose is locked in the N configuration of type in the C3, has formed inflexible condensation structure.LNA has advantage such as nontoxicity in the hybridization avidity powerful with DNA/RNA, antisense activity, nuclease-resistant ability, good water solubility and the body as a kind of new antisense nucleic acid.Can prepare the LNA probe thereby it can combine specifically with DNA and RNA, compare with dna probe, the stability of its hybridization and specificity increase, and can improve the efficient and the sensitivity of gene diagnosis greatly.The present invention designs lock nucleic acid retardance probe, its sequence and wild template are mated fully, part base lock nucleination, be used for the KRAS wild-type DNA of specificity in conjunction with template DNA, because of its terminal phosphateization, thereby can't extend the non-specific amplification that suppresses the wild-type template, increase sensitivity and the specificity that sudden change detects greatly.
Amplification refractory mutation system (amplification refractory mutation system ARMS) was set up in 1989, was used for the known mutations gene is detected.This method is by design sudden change specificity ARMS primer, and the mutating alkali yl coupling of its 3 ' terminal bases and mutant to be detected is used for specific amplification sudden change template.The detection sensitivity of ARMS depends on specificity and the reaction conditions such as the enzyme of ARMS primer, the optimization of magnesium ion concentration etc.In order to increase the specificity of primer, reduce primer and extend with the mispairing of the wrong timing of target DNA, can be by introducing another one or two base mismatch in 2-3 base of primer 3 ' end, make it and template between the multiple mispairing of formation to stop the mistake extension.
The present invention will lock the wild retardance probe of nucleic acid (LNA) and ARMS sudden change Auele Specific Primer combines, and is used for the KRAS gene mutation typing.This detection kit designs at the detection of KRAS gene hot saltation zone, comprises totally 7 kinds of mutator gene types of KRAS gene the 12nd, 13 codons.This test kit comprises the 25ul qPCR reaction system through optimizing, the feature of this reaction system is all to contain the qPCR mixed reaction solution, the LNA retardance sequence of TaqMan probe positive reference substance and the amplification of inhibition wild-type KRAS genomic dna, just the ARMS primer is distinguished to some extent at special mutational site.
Probe used in the present invention is 5 ' the TaqMan probe of end FAM mark, the oligonucleotide of probe two ends difference mark fluorescent reporter group (R) and fluorescent quenching group (Q).When probe was complete, when promptly random state and no PCR product were hybridized state, the fluorescence that reporter group sends was absorbed by quenching group.In the ARMS-qPCR amplification procedure, 5 ' of Goldstarbest Taq enzyme end 5 prime excision enzyme activity is simultaneously also the probe cracking when special PCR product and TaqMan probe generation hybridization, and the photofluorometer that the fluorescence that reporter group discharged just can be built in the detection by quantitative instrument detects.PCR is every through a circulation, and fluorescent signal is also the same with the purpose fragment, and the process that has sync index to increase, the power of fluorescent signal have just been represented what of copy number of template DNA.Therefore the present invention not only can be used for simple qualitative detection, also can be used as the detection by quantitative of the concrete content of sample.
The advantage of comprehensive ARMS primer and LNA retardance probe, and the characteristics of real-time fluorescence quantitative PCR technology, with sudden change detection technique comparison such as direct order-checking, the present invention is used to detect the KRAS transgenation and has following advantage:
1. high specificity: the ARMS primer of design is respectively at seven kinds of special mutant nucleotide sequences of KRAS gene the 12nd and the 13rd codon, can the corresponding sudden change template DNA of specific amplification; 3 ' terminal 2-3 reciprocal position at the ARMS primer increases one or two base mispairings, can increase the specificity of ARMS primer; The technology of the present invention adds the lock nucleic acid retardance probe of wild-type KRAS gene specific in the PCR reaction system, thereby by suppressing the amplification of wild type gene group DNA cloning enrichment mutant DNA, has further improved the specificity that detects.
2. susceptibility height: the technology of the present invention adds wild-type KRAS gene lock nucleic acid retardance probe (LNA) in the PCR reaction solution, by suppressing the amplification of wild-type template specifically, thereby reaches the inrichment to trace sudden change template, improves detection sensitivity; Present technique detects KRAS transgenation susceptibility and reaches 0.1-1% (being that the 0.1-1% that mutator gene group DNA reaches the total DNA of gene can detect); And direct sequencing detects the susceptibility of KRAS sudden change and is about 10% (be mutator gene group DNA reach 10% of the total DNA of gene just can detect).
3. testing process is the stopped pipe reaction, greatly reduces the possibility of pollution and result error.
4. simple to operate fast, inspect by ready samples to from sample and to obtain a result and can finish with interior at 3 hours.And direct sequencing detects complex steps: censorship sample → extraction DNA → pcr amplification → checking PCR product (electrophoresis) → purified pcr product → directly order-checking, and the electrophoresis process of two PCR products of its experience, opportunities for contamination is big, is not suitable for carrying out on a large scale in hospital.
5. interpretation as a result is clear and definite, objective; Also can carry out quantitative analysis if need to the result.
6. high-throughput once can detect 48 examples at most.
7. safety: do not comprise hazardous and noxious substances in the whole system, need not the aftertreatment of PCR product, operator and environment are not had harm.
Description of drawings
Fig. 1 is for detecting the amplification curve diagram of KRAS gene the 12nd codon GGT → GAT base substitution mutation of colorectal cancer positive sample with the ARMS-qPCR detection kit, it is negative that this sample confirms that through order-checking GGT → GAT suddenlys change, it is positive to confirm that through the T-A clone GGT → GAT suddenlys change, and the positive colony rate is about 3%;
Fig. 2 is for detecting the amplification curve diagram of KRAS gene the 12nd codon GGT → GTT base substitution mutation of colorectal cancer positive sample with the ARMS-qPCR detection kit, it is positive that this sample confirms that through order-checking GGT → GTT suddenlys change;
Fig. 3 is for detecting the amplification curve diagram of KRAS gene the 12nd codon GGT → GCT base substitution mutation of colorectal cancer positive sample with the ARMS-qPCR detection kit, it is positive that this sample confirms that through order-checking GGT → GCT suddenlys change;
Fig. 4 is for detecting the amplification curve diagram of KRAS gene the 12nd codon GGT → AGT base substitution mutation of intestinal cancer positive sample with the ARMS-qPCR detection kit, it is negative that this sample confirms that through order-checking GGT → AGT suddenlys change, it is positive to confirm that through the T-A clone GGT → AGT suddenlys change, and the positive colony rate is about 6%;
Fig. 5 is for detecting the amplification curve diagram of KRAS gene the 12nd codon GGT → TGT base substitution mutation of intestinal cancer positive sample with the ARMS-qPCR detection kit;
Fig. 6 is the amplification curve diagram of KRAS gene the 12nd codon GGT → CGT base substitution mutation for detecting positive control 12Arg with the ARMS-qPCR detection kit, and this mutant form is relatively more rare in Chinese knot rectum cancer crowd.
Fig. 7 is for detecting the amplification curve diagram of KRAS gene the 13rd codon GGT → GAT base substitution mutation of intestinal cancer positive sample with the ARMS-qPCR detection kit;
Fig. 8 is for detecting the amplification curve diagram of the KRAS gene wild-type of intestinal cancer positive sample with the ARMS-qPCR detection kit.
Embodiment
For making the present invention easier to understand, will further set forth concrete case study on implementation of the present invention below.
Collect the wax stone tissue that clinical pathology is diagnosed as 100 routine patients of knot rectal adenocarcinoma (CRC), all do not accept Cetuximab (cetuximab) treatment before all patient's arts.Experimental applications under the extraction genomic dna is provided with from wax stone.Detect the common 7 kinds of base substitution mutations of KRAS gene the 12nd, 13 codon with ARMS-qPCR.
Design and screening can specific detection above-mentioned 7 kinds of base substitution mutations each one of specificity ARMS primer and with reference to one of primer, design one of general downstream primer, design is also screened one of general TaqMan probe, one of probe is blocked in design also screening LNA, and each probe, primer sequence are distinguished specific as follows:
The LNA sequence is shown in SEQ ID NO A0 in the sequence table: 5 '-TGGAGCTG
GTG
GCGTAGGC-PO4-3 '.
General downstream primer sequence is: SEQ ID NO A1:5 '-ACCTCTATTGTTGGATCATATTCGTC-3 '
The TaqMan probe sequence is: SEQ ID NO A2:5 '-FAM-GAATTAGCTGTATCGTCAAGGCACTCT-3 '
With reference to primer sequence be: SEQ ID NO A3:5 '-CTGAATATAAACTTGTGGTAGTT-3 '
At KRAS gene the 12nd codon GGT → GAT
The ARMS primer sequence of mutant is: SEQID NO A4:5 '-CTTGTGGTAGTTGGAGCTTA-3 '
At KRAS gene the 12nd codon GGT → GTT
The ARMS primer sequence of mutant is: SEQID NO A5:5 '-AAACTTGTGGTAGTTGGAGCGGT-3 '
At KRAS gene the 12nd codon GGT → GCT
The ARMS primer sequence of mutant is: SEQID NO A6:5 '-AACTTGTGGTAGTTGGAGCTGC-3 '
At KRAS gene the 12nd codon GGT → AGT
The ARMS primer sequence of mutant is: SEQID NO A7:5 '-ATAAACTTGTGGTAGTTGGAGCTA-3 '
At KRAS gene the 12nd codon GGT → TGT
The ARMS primer sequence of mutant is: SEQID NO A8:5 '-AACTTGTGGTAGTTGGAGCGT-3 '
At KRAS gene the 12nd codon GGT → CGT
The ARMS primer sequence of mutant is: SEQID NO A9:5 '-ATAAACTTGTGGTAGTTGGAGCCC-3 '
At KRAS gene the 13rd codon GGC → GAC
The ARMS primer sequence of mutant is: SEQID NO A10:13Asp-3:5 '-GTGGTAGTTGGAGCTGGTAA-3 '
1. the optimization of reaction system:
(1) under the optimization of the primer concentration situation that other condition is identical in reaction system, primer concentration being done the multiple proportions serial dilution from 0.1 μ mol/L to 1.6 μ mol/L respectively, is 0.3 μ mol/L by the analysis of test-results is relatively decided best primer concentration.
(2) under the optimization of the concentration and probe concentration situation that other condition is identical in reaction system, with concentration and probe concentration respectively from, true 0.05 μ mol/L to 0.5 μ mol/L does the multiple proportions serial dilution, compares by the analysis to test-results, determines that best concentration and probe concentration is 0.1 μ mol/L.
(3) under the optimization of the annealing temperature situation that other condition is identical in reaction system, carry out grads PCR (56 ° to 64 ° annealing temperatures), compare, determine that optimum annealing temperature is 60 ° by analysis to test-results.
(4) optimization of enzyme adds various enzymes in reaction system, compares by the analysis to test-results, determines that best enzyme is a gold star TAQ enzyme.
Through optimizing reaction system is 25 μ l, comprises qPCR mix 20 μ l, and with reference to primer or each 0.75 μ l (final concentration 0.3 μ M) of ARMS primer, LNA blocks probe 2.25 μ l (final concentration 0.9 μ M) template DNA 2.0 μ l (final concentration 10-300ng/ μ l).Described qPCR mix component and final concentration thereof are:
PCR damping fluid final concentration is 1 *;
The dNTPs final concentration is 0.2mM;
General downstream primer final concentration is 0.3 μ M;
TaqMan probe final concentration is 0.1 μ M;
Goldstarbest Taq enzyme final concentration is 0.05U/ μ L;
MgCl
2Final concentration is 3.5mM;
ARMS-qPCR carries out on the ABI7500 detector.Each sample carries out 8 tube reactions, and every tube reaction adds common qPCR mix, LNA retardance probe and template DNA, and different has only with reference to primer or ARMS primer.The PCR reaction conditions is: 95 ℃ of pre-sex change 10 minutes, 95 ℃ 15 seconds, 60 ℃ 40 seconds, amplified reaction 40 circulations are collected fluorescence 60 ℃ of 40 second stages.
The result is: detect 20 routine samples altogether in 100 routine samples KRAS gene the 12nd codon GGT → GAT sudden change takes place, KRAS gene the 12nd codon GGT → GTT sudden change takes place in 4 routine samples, KRAS gene the 12nd codon GGT → GCT sudden change takes place in 2 routine samples, KRAS gene the 12nd codon GGT → AGT sudden change takes place in 3 routine samples, KRAS gene the 13rd codon GGC → GAC sudden change takes place in 11 routine samples, and KRAS gene the 12nd codon GGT → TGT sudden change takes place 2 routine samples.Wherein 3 routine samples detect interpretation through the ARMS-qPCR of KRAS gene mutation typing to be that the 12nd codon GGT → GAT suddenly change positive, and it is negative to be through sequencing result that GGT → GAT suddenly change, and through T-A clone confirmation GGT → GAT positive of suddenling change, the positive colony rate is about 3-7%; Wherein to detect interpretation be the 12nd codon GGT → positive this sample of AGT sudden change to 1 routine sample KRAS gene type ARMS-qPCR that to be through sequencing result that GGT → AGT suddenly change negative, confirms GGT → AGT positive of suddenling change through the T-A clone, and the positive colony rate is about 6%.All the other results all are consistent with direct sequencing result.
It is highly sensitive that The above results shows that ARMS-qPCR method of the present invention detects tumor tissues KRAS transgenation, can detect the trace sudden change template (＜10%) in the clinical sample; Its reliable results detects " gold standard " with sudden change: direct Ce Xu coincidence rate as a result 〉=95%.Described in addition method is quick, simple to operate, and interpretation as a result is objective, closes the border the reaction pollution and lacks, and is very suitable for carrying out on a large scale in clinical.
Claims (1)
1. be used for the ARMS-qPCR detection kit of KRAS gene mutation typing, it is characterized in that, this test kit comprises qPCR mixed reaction solution, lock nucleic acid retardance probe, with reference to primer, ARMS primer and positive control sample; Wherein the qPCR mixed reaction solution comprises: PCR damping fluid, dNTPs, MgCl
2, Goldstarbest Taq enzyme, the general downstream primer of PCR and general TaqMan probe;
The primer of ARMS described in the test kit comprises seven kinds of upstream primers, is used for the corresponding respectively KRAS of detection gene the 12nd codon GAT, GTT, GCT, TGT, AGT and these six kinds of mutants of CGT and No. 13 this a kind of mutation type of codon GAC; The sequence of these seven kinds of upstream primers is shown in SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11;
The qPCR reaction system is 25 μ l, and the final concentration of each component is respectively: the PCR damping fluid is 1 *; DNTPs is 0.01～1.5mM; MgCl
2Be 0.5～5mM; Goldstarbest Taq enzyme is 0.05U/ μ L; The general downstream primer of PCR is 0.1～1 μ M; General TaqMan probe is 0.1～1 μ M; Lock nucleic acid retardance probe is 0.9 μ M, be that 0.3 μ M, ARMS primer are that 0.3 μ M, positive control sample are 10～300ng/ μ l with reference to primer;
The sequence of described lock nucleic acid retardance probe is shown in SEQ ID NO 1;
The sequence of the general downstream primer of described PCR is shown in SEQ ID NO 2;
The sequence of described general TaqMan probe is shown in SEQ ID NO 3;
Described is the upstream primer of can increase simultaneously a KRAS wild-type and a mutated genes group DNA with reference to primer, and its sequence is shown in SEQ ID NO 4;
ARMS primer described in the qPCR reaction system is any one in seven kinds of upstream primers, is used for the corresponding respectively KRAS of detection gene the 12nd codon GAT, GTT, GCT, TGT, AGT and these six kinds of mutants of CGT and No. 13 this a kind of mutation type of codon GAC; The sequence of these seven kinds of upstream primers is shown in SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11;
Described positive control sample is the plasmon DNA that has aforementioned KRAS genic mutation type respectively.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110327916 CN102367478B (en) | 2011-10-25 | 2011-10-25 | ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110327916 CN102367478B (en) | 2011-10-25 | 2011-10-25 | ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102367478A CN102367478A (en) | 2012-03-07 |
| CN102367478B true CN102367478B (en) | 2013-07-31 |
Family
ID=45760068
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110327916 Expired - Fee Related CN102367478B (en) | 2011-10-25 | 2011-10-25 | ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102367478B (en) |
Families Citing this family (31)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102888466A (en) * | 2012-10-30 | 2013-01-23 | 武汉友芝友生物制药有限公司 | KRAS gene mutation detection kit and detection method |
| CN103215361A (en) * | 2013-04-18 | 2013-07-24 | 深圳联合医学科技有限公司 | Allele variant detection method, kit and composition |
| CN103436613B (en) * | 2013-08-21 | 2015-10-07 | 中国人民解放军第二军医大学 | Fluorescence PCR detection reagent kit of IDH1/IDH2 transgenation and uses thereof |
| CN104164491A (en) * | 2014-07-21 | 2014-11-26 | 北京新基永康生物科技有限公司 | ARMS-qPCR detection method and kit for helicobacter pylori 23S rDNA gene mutation subtype |
| CN105886599A (en) * | 2014-10-20 | 2016-08-24 | 江苏所罗门兄弟医学科技有限公司 | ARMS-qPCR detection kit and detection method for ABCB1 genotyping |
| WO2016103727A1 (en) * | 2014-12-25 | 2016-06-30 | 凸版印刷株式会社 | Method for promoting amplification of specific nucleic acid sequence |
| CN104531881A (en) * | 2015-01-09 | 2015-04-22 | 湖南圣湘生物科技有限公司 | Fluorescence PCR detection kit for human K-RAS gene mutation |
| CN104830981A (en) * | 2015-04-29 | 2015-08-12 | 广州和实生物技术有限公司 | KRAS gene multipoint mutation single tube rapid detection method and kit |
| CN104805208B (en) * | 2015-04-30 | 2017-12-05 | 山东维真生物科技有限公司 | For detecting the primer combination of probe thing, kit and detection method of 7 kinds of hot spot mutations of mankind KRAS genes |
| CN104862401B (en) * | 2015-05-29 | 2017-10-31 | 沈阳优吉诺生物科技有限公司 | Detect primer, kit and its PCR method of KRAS gene hot mutant sites |
| CN105567837B (en) * | 2016-02-02 | 2019-01-29 | 河南中医学院第一附属医院 | Detect primer and probe system, method and the kit of K-ras gene mutation |
| CN108330191A (en) * | 2017-01-20 | 2018-07-27 | 江苏众红生物工程创药研究院有限公司 | Primer, probe and kit for the detection of people's KRAS gene mutation |
| CN106755489A (en) * | 2017-01-22 | 2017-05-31 | 杭州金溪生物技术有限公司 | High sensitivity detection method of gene mutation and used kit |
| CN106868127B (en) * | 2017-02-20 | 2019-09-10 | 人和未来生物科技（长沙）有限公司 | People's KRAS gene multipoint mutation single tube joint inspection fluorescence PCR method, kit and system |
| CN106755551A (en) * | 2017-03-20 | 2017-05-31 | 陈超 | A kind of EGFR/L858R is mutated liquid biopsy kit and its application |
| CN106987622A (en) * | 2017-03-20 | 2017-07-28 | 杭州迪安医学检验中心有限公司 | Pyrosequencing joint amplification block technique detection KRAS genes 12 and 13 codon low frequency mutant primers and its application |
| CN107604053A (en) * | 2017-08-16 | 2018-01-19 | 金绍莲 | Application and its kit and detection method of a kind of wild-type amplification blocker in detection gene trace mutation reagent is prepared |
| CN107385066B (en) * | 2017-08-17 | 2021-03-23 | 浙江大学 | Primer pair, kit, detection method and application for detecting EGFR gene mutation |
| CN107447013B (en) * | 2017-08-31 | 2020-06-05 | 上海伯豪生物技术有限公司 | Method for detecting mutation sites of codons 12 and 13 of Kras gene and kit thereof |
| CN108048531B (en) * | 2017-11-16 | 2021-06-18 | 苏州吉玛基因股份有限公司 | A high-sensitivity ultra-blocking fluorescence quantitative PCR method for the detection of rare mutations |
| CN108060215A (en) * | 2018-02-08 | 2018-05-22 | 天津安必森生物技术有限公司 | A kind of kit for detecting people's KRAS gene mutation |
| CN108220444A (en) * | 2018-02-28 | 2018-06-29 | 无锡禾盛医疗器械有限公司 | A kind of primer combination of probe of KRAS gene mutation detection and its application |
| CN109136345A (en) * | 2018-09-21 | 2019-01-04 | 北京知光基因科技有限公司 | A kind of PCR method and its application expanded and detect low-content gene mutation |
| CN109439756A (en) * | 2018-11-30 | 2019-03-08 | 广东腾飞基因科技股份有限公司 | Kit for detecting KRAS related drug resistance |
| CN110592217A (en) * | 2019-10-08 | 2019-12-20 | 湖南艾佳生物科技股份有限公司 | Kit for detecting KRAS gene mutation in free DNA of peripheral blood and application thereof |
| CN110982898A (en) * | 2019-10-14 | 2020-04-10 | 苏州艾可瑞斯生物科技有限公司 | MyD88 gene L265P mutation detection kit based on ARMS-PCR and detection method |
| CN111118119B (en) * | 2019-12-18 | 2023-09-26 | 杭州瑞普基因科技有限公司 | Method for enriching and detecting target mutations using base mismatch blockers |
| CN110982886A (en) * | 2019-12-30 | 2020-04-10 | 武汉光谷联合医学检验所股份有限公司 | Human K-ras gene mutation detection kit and application thereof |
| CN110964833B (en) * | 2020-01-06 | 2023-05-02 | 中南大学湘雅医院 | A kit for detecting KRAS and BRAF gene mutations in plasma cell-free DNA in one tube |
| CN112322739B (en) * | 2020-11-24 | 2023-08-15 | 深圳市罗湖区人民医院 | Method and kit for detecting ADGRG6 high-frequency mutation site |
| CN115927637A (en) * | 2022-12-20 | 2023-04-07 | 苏州普熙生生物医药科技有限公司 | Primer group, kit and detection method for KRAS gene mutation detection |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101608241A (en) * | 2009-04-13 | 2009-12-23 | 厦门艾德生物医药科技有限公司 | Be used to detect primer, probe and the using method thereof of human K-ras transgenation |
| WO2011104695A2 (en) * | 2010-02-26 | 2011-09-01 | GAMMAGENETICS Sàrl | Detection of kras mutation in exon 2 by allele specific real time quantitative pcr (as-qpcr) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2376659B1 (en) * | 2008-12-17 | 2015-12-02 | Life Technologies Corporation | Methods, compositions, and kits for detecting allelic variants |
-
2011
- 2011-10-25 CN CN 201110327916 patent/CN102367478B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101608241A (en) * | 2009-04-13 | 2009-12-23 | 厦门艾德生物医药科技有限公司 | Be used to detect primer, probe and the using method thereof of human K-ras transgenation |
| WO2011104695A2 (en) * | 2010-02-26 | 2011-09-01 | GAMMAGENETICS Sàrl | Detection of kras mutation in exon 2 by allele specific real time quantitative pcr (as-qpcr) |
Non-Patent Citations (4)
| Title |
|---|
| High sensitive mutation analysis on KRAS gene using LNA/DNA chimeras as PCR amplification blockers of wild-type alleles;Qing Huang, et al.;《Molecular and Cellular Probes》;20100821;376-380 * |
| Qing Huang, et al..High sensitive mutation analysis on KRAS gene using LNA/DNA chimeras as PCR amplification blockers of wild-type alleles.《Molecular and Cellular Probes》.2010,376-380. |
| Reid P Oldenburg, et al..Selective Amplification of Rare Mutations Using Locked Nucleic Acid Oligonucleotides that Competitively Inhibit Primer Binding to Wild-Type DNA.《Journal of Investigative Dermatology》.2007,第128卷398-402. |
| Selective Amplification of Rare Mutations Using Locked Nucleic Acid Oligonucleotides that Competitively Inhibit Primer Binding to Wild-Type DNA;Reid P Oldenburg, et al.;《Journal of Investigative Dermatology》;20070621;第128卷;398-402 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102367478A (en) | 2012-03-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102367478B (en) | ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and detection method | |
| JP7384459B2 (en) | Detection technology system and use for enriching low abundance DNA mutations based on nuclease cooperative PCR principles | |
| CN107419018B (en) | Method and kit for detecting gene mutation based on Blocker primer and ARMS primer | |
| CN104946739B (en) | EGFR genetic mutation detection kit and its application | |
| CN103382503B (en) | EGFR gene 19 exon 19 kinds of deletion mutantion detection kit and detection method | |
| CN110117653A (en) | The detection method and kit of the mutation rate in lung cancer mutational site | |
| EP1809770A2 (en) | Molecular analysis of cellular fluid and liquid cytology specimens for clinical diagnosis, characterization, and integration with microscopic pathology evaluation | |
| US10689709B2 (en) | Kit and method for detecting mutations in CTNNB1 and hTERT, and use thereof in HCC detection and disease management | |
| CN101899496A (en) | K-Ras gene mutation typing fluorescence quantitative PCR detection kit and detection method thereof | |
| CN104805207A (en) | Kit for detecting kras gene mutation, and detection method of kit | |
| CN114672568B (en) | Kit for detecting cervical cell gene methylation | |
| CN110527710B (en) | Primer, probe and kit for detecting NTRK gene fusion mutation | |
| US20120237935A1 (en) | Reagent kit for quantitatively detecting the mutations of epidermal growth factor receptor(egfr) | |
| CN102220413A (en) | Method for detecting exon 19 deletion mutation and exon 21 point mutation of epidermal growth factor receptor gene | |
| CN106148497A (en) | BRAF gene mutation detection kit and application thereof | |
| WO2011079524A1 (en) | Kits for quantitative detection of k-ras mutations | |
| CN104152556A (en) | Kit for judging adjunctive therapeutic effects of trastuzumab on breast cancer and application | |
| CN113122629B (en) | Primer and probe for hairpin amplification blocking method for gene mutation detection and application thereof | |
| CN108130362A (en) | Kit and application for EGFR genetic mutation detection | |
| CN110964833B (en) | A kit for detecting KRAS and BRAF gene mutations in plasma cell-free DNA in one tube | |
| CN105734137A (en) | Probe method for detecting human H3F3A gene mutation and detection kit | |
| CN116287255A (en) | Pancreatic cancer diagnosis kit | |
| CN105506140B (en) | ROS1 fusion ARMS fluorescence quantitive PCR typing detection kit | |
| CN103397102A (en) | Detection method for quantifying gene mutation abundance | |
| CN116732171B (en) | Primer probe combination for screening colorectal cancer methylation double sites and kit thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130731 Termination date: 20191025 |