CN109306379A - For detecting primer, detection method and the kit of human EGFR gene T790M mutation - Google Patents
For detecting primer, detection method and the kit of human EGFR gene T790M mutation Download PDFInfo
- Publication number
- CN109306379A CN109306379A CN201811044900.0A CN201811044900A CN109306379A CN 109306379 A CN109306379 A CN 109306379A CN 201811044900 A CN201811044900 A CN 201811044900A CN 109306379 A CN109306379 A CN 109306379A
- Authority
- CN
- China
- Prior art keywords
- mutation
- gene
- primer
- human egfr
- egfr
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides primer, detection method and the kits for detecting human EGFR gene T790M mutation.Detection primer provided by the invention can expand mutant nucleotide sequence in conjunction with EGFR gene conserved sequence.Detection method provided by the invention can issue fluorescence signal using detection fluorescence signal group in conjunction with amplified fragments, specifically bound using blocking agent wild-type sequence corresponding with mutational site, inhibit wild type non-specific amplification.The present invention establishes the kit based on real-time fluorescence PCR using competitive ApoE gene amplification technique, can accurately detect EGFR gene T790M mutation.The method of the present invention is easy to operate, as a result readable, and high sensitivity can detect the sample of the T790M of gene containing 0.001%EGFR mutation, realizes the super sensitivity detection being mutated to EGFR gene T790M.
Description
This application claims on October 13rd, 2017 to submit Patent Office of the People's Republic of China, application No. is 201710955204.4, invention name
Chinese patent application referred to as " for the primer of super sensitivity detection human EGFR gene mutations, detection method and kit " it is excellent
It first weighs, entire contents are hereby incorporated by reference in the application.
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of for detecting drawing for human EGFR gene T790M mutation
Object, detection method and kit.
Background technique
Human epidermal growth factor acceptor (epidermal growth factor receptor, EGFR) is a kind of albumen
Tyrosine kinase receptor is distributed widely in the cells such as mammalian epithelial cell, fibroblast, spongiocyte, horn cell
Surface has tyrosine kinase activity.It is one of HER/ErbB family member, therefore also known as HER1 or ErbB1.EGFR and its
Dimer is formed in cell surface after ligand binding, by the activity of tyrosine kinase, activated receptor autophosphorylation, in the cell
Signal is issued, by the cascade reaction of adaptin in cytoplasm and enzyme, adjusts the transcription of transcription factor activation gene, guidance is thin
Born of the same parents migrate, stick, being proliferated, breaking up and apoptosis.When EGFR mutates, EGFR itself or its ligand are overexpressed, to pass through
Autocrine or paracrine mode stimulate cell to form uncontrolled proliferation.In addition, EGFR overactivity can also start multiple protein
The expression of hydrolase and angiogenic factors, to accelerate the transfer of cancer cell.Therefore, the usual prognosis of EGFR overexpression person compared with
Difference.
U.S. Food and Drug Administration has approved U.S.'s AstraZeneca (Aotrazeneca) company in May, 2003
The small molecule gene target drug Gefitinib (Gefitinib, also referred to as Iressa/Iressa) of production is non-for treating advanced stage
Small Cell Lung Cancer (NSCLC), China also had approved Gefitinib with 2 months 2005 and enter clinic.Gefitinib is a kind of oral
Small molecule EGFR tyrosine kinase inhibitor (EGFR-TKI), it can specifically competitive binding cancer cell EGFR kinases function
The ATP-binding site in energy area inhibits growth, the proliferation of tumour by inhibiting the activity of the enzyme, but makees without apparent chemotherapy pair
With.Researcher Lynch of Harvard Medical School of the U.S. etc. takes the lead in reporting have EGFR tyrosine kinase gene coding in lung carcinoma cell
The patient of 18~21 exons mutation of area, the effective percentage of targeted drug Gefitinib are up to 80% or more.This phenomenon may be
Since EGFR mutation reconstructs certain groups of EGFR tyrosine kinase A TP binding site, enhance and ATP or its is competing
The interaction of striving property inhibitor Gefitinib.Therefore, the patient of EGFR genetic mutation shows better to Gefitinib
Therapeutic response.Studies have shown that only have about 10% to have EGFR genetic mutation in U.S.'s Patients with Non-small-cell Lung, however in Asian
In, about 30% Patients with Non-small-cell Lung has EGFR genetic mutation.Clinical application the result shows that, EGFR gene extron
18~21 mutation (somatic mutation) is patient to the effective prerequisite of such targeted drug, if patient does not carry
EGFR genetic mutation and take such drug, not only delay the waste that the state of an illness also causes huge expenses for medicine.Therefore, the screening before medication
Whether detection patient, which carries EGFR genetic mutation, is particularly important and the developing direction of future tumors individualized treatment.
Currently, for EGFR genetic mutation detection method mainly have direct sequencing, traditional fluorescence quantitative PCR method and
High-resolution melting curve method.Time-consuming and sensitivity is low for PCR sequencing PCR, is generally only used for scientific research, it is difficult to for clinical detection.
Traditional fluorescence quantitative PCR method is difficult to the amplification for making mutated genes obtain specificity in a large amount of wild type gene, is easy
Generate the testing result of false positive.Instrument used in high-resolution melting curve method is more special, it is difficult to it is popularized in hospital,
And the method, when detecting deletion mutation, effect is poor.
It is early to find early treatment if oncogene can be gone out in cancer early detection, in addition, quick and precisely delicately detect these with
The related gene mutation of drug responsiveness, can greatly improve the survival rate of cancer patient.But due to cancer early carcinomatous change sample
Oncogene content especially in blood sample is seldom, and the detection sensitivity of above method at most can only achieve 0.2%, much
Do not reach the requirement that extremely micro abnormal DNA molecular is detected under a large amount of normal DNA backgrounds also, it is therefore, quick and precisely sensitive
Ground detects these EGFR genetic mutations related with drug responsiveness, just becomes people's urgent problem to be solved.
Summary of the invention
In view of this, the present invention provides the primers for detecting human EGFR gene T790M mutation, detection method.This
Invention additionally provides the kit containing the primer and detection method, by specific amplification technology, is inhibiting normal background
In the case of, micro abnormal DNA molecular exponential amplification, one can be detected under the background of 100,000 normal DNA molecules
Abnormal DNA molecular, sensitivity reach 0.001%, realize cancer early detection.
In order to achieve the above objectives, first aspect present invention provides the detection for detecting human EGFR gene T790M mutation
Method, comprising the following steps: will be used to detect the primer, blocking agent, detection fluorescence signal base of human EGFR gene T790M mutation
Group, the mixing of testing gene sample, carry out real-time fluorescence quantitative PCR, obtain testing result；
Wherein, described for detecting under the primer of human EGFR gene T790M mutation, including mutation upstream primer and mutation
Primer is swum, mutation upstream primer includes the primer that 1-10 item is directed to different mutational sites, close the length is 15-40 base
1st, 2,3,4 or 5 base of 3 ' ends is mutational site, complementary with a chain of EGFR mutated genes to be measured；Mutation
Downstream primer is a shared downstream primer, and the length is 15-40 bases, another with EGFR mutated genes to be measured
Chain is complementary；
The blocking agent can inhibit the non-spy of wild type with the mutational site EGFR gene T790M wild-type sequence specific bond
Different amplification；
The detection fluorescence signal group in conjunction with pcr amplified fragment and can issue fluorescence signal.
In an embodiment of the present invention, the mutation upstream primer is directed to the primer in mutational site, length including 1
For 33 bases, wherein the 3rd bit base of 3 ' ends is mutational site, it is complementary with a chain of EGFR mutated genes to be measured.
In an embodiment of the present invention, the mutation downstream primer, the length is 24 bases, with EGFR saltant type to be measured
Another chain complementation of gene.
In a preferred embodiment, described for detecting the primer of human EGFR gene T790M mutation, sequence
Include:
It is mutated upstream primer: TCTGCCTCACCTCCACCGTGCAGCTCATCATGC (SEQ ID NO.1), is mutated downstream
Primer: AATATTGTCTTTGTGTTCCCGGAC (SEQ ID NO.2).
In an embodiment of the present invention, the nucleic acid sequence of the blocking agent matching EGFR wild type gene, the nucleic acid sequence
Column are modified with chemical group.
In a preferred embodiment, the nucleic acid sequence of matching EGFR wild type gene includes: in the blocking agent
AGCTCATCACGCAGCTCAT(SEQ ID NO.3)；The chemical group includes: amido, phosphate group, biotin, biology
One of element-TEG and C3-spacer or a variety of.
In an embodiment of the present invention, the detection fluorescence signal group include: FAM, Cy3, HEX, Texas Red,
One of JOE, VIC, TAMRA, Cy5, SYTO 9, SYBR MIX or a variety of.
Second aspect of the present invention provides the primer for detecting human EGFR gene T790M mutation, including mutation upstream is drawn
Object and mutation downstream primer, mutation upstream primer include the primer that 1-10 item is directed to different mutational sites, and the length is 15-40
Base, the 1st, 2,3,4 or 5 base close to 3 ' ends are mutational site, a chain with EGFR mutated genes to be measured
It is complementary；Mutation downstream primer is a shared downstream primer, and the length is 15-40 bases, with EGFR saltant type base to be measured
Another chain complementation of cause.
In an embodiment of the present invention, the mutation upstream primer is directed to the primer in mutational site, length including 1
For 33 bases, wherein the 3rd bit base of 3 ' ends is mutational site, it is complementary with a chain of EGFR mutated genes to be measured.
In an embodiment of the present invention, the mutation downstream primer, the length is 24 bases, with EGFR saltant type to be measured
Another chain complementation of gene.
In a preferred embodiment, described for detecting the primer of human EGFR gene T790M mutation, sequence
Include:
It is mutated upstream primer: TCTGCCTCACCTCCACCGTGCAGCTCATCATGC (SEQ ID NO.1), is mutated downstream
Primer: AATATTGTCTTTGTGTTCCCGGAC (SEQ ID NO.2).
Third aspect present invention provides the blocking agent for detecting human EGFR gene T790M mutation, the blocking agent energy
With EGFR gene wild-type sequence specific bond, inhibit wild type non-specific amplification.
In an embodiment of the present invention, the nucleic acid sequence of the blocking agent matching EGFR wild type gene, the nucleic acid sequence
Column are modified with chemical group.
In a preferred embodiment, the nucleic acid sequence of matching EGFR wild type gene includes: in the blocking agent
AGCTCATCACGCAGCTCAT(SEQ ID NO.3)；The chemical group includes: amido, phosphate group, biotin, biology
One of element-TEG and C3-spacer or a variety of.
Fourth aspect present invention provides the kit for detecting human EGFR gene T790M mutation, including such as the present invention
For detecting the primer of human EGFR gene T790M mutation, as described in the third aspect of the present invention being used for described in second aspect
Detect the blocking agent and detection fluorescence signal group of human EGFR gene T790M mutation.
In an embodiment of the present invention, the detection fluorescence signal group include: FAM, Cy3, HEX, Texas Red,
One of JOE, VIC, TAMRA, Cy5, SYTO 9, SYBR MIX or a variety of.
Fifth aspect present invention provides the human EGFR gene T790M that is used to detect as described in the first aspect of the invention and is mutated
The detection method, primer for detecting human EGFR gene T790M mutation as described in respect of the second aspect of the invention, such as of the invention
For detecting the blocking agent of human EGFR gene T790M mutation and/or as described in fourth aspect present invention described in the third aspect
For detect human EGFR gene T790M mutation kit detection human EGFR gene mutations in application.
In an embodiment of the present invention, in the application, in the tested sample, the concentration of the mutated genes is not
Lower than 1%, 0.1%, 0.01% or 0.001%.
Further, the mutated genes concentration is the ratio that modification gene accounts for wild type gene quantity or molar concentration
Example.
Further, the mutated genes concentration is the ratio that modification gene accounts for the total gene dosage of sample or molar concentration
Example.
It can be normal wild at 100,000 provided by the present invention for the detection method of detection human EGFR gene T790M mutation
In type DNA molecular, detects existing 1 abnormal sudden change type DNA molecular, the gene mutation containing 0.001%EGFR can be detected
Sample realizes cancer early detection,.
Detailed description of the invention
Fig. 1 is schematic illustration provided in an embodiment of the present invention；
Fig. 2 is provided in an embodiment of the present invention plus blocking agent system and blocking agent system expanding effect comparison diagram is not added,
Middle A1 is the amplification curve that blocking agent system amplification mutated genes are not added, B1 is that blocking agent system is not added to expand wild type base
Amplification curve, the A2 of cause are plus amplification curve, the B2 of blocking agent system amplification mutated genes are to add the amplification of blocking agent system wild
The amplification curve of raw type gene；
Fig. 3 is the sensitivity technique of kit provided in an embodiment of the present invention, and wherein A, B, C, D are respectively that mutated gene is dense
The amplification curve that degree is 1%, 0.1%, 0.01%, 0.001%；
Fig. 4 is the specific detection of kit provided in an embodiment of the present invention, and wherein A is to Sudden Anomalies gene magnification, B
To be expanded to normal gene, C be to TP53 gene magnification, D be KIT gene magnification, E is to expand to non-targeted gene compound.
Specific embodiment
The following is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as
Protection scope of the present invention.
Below in conjunction with Fig. 1, a preferred embodiment of the present invention will be described in detail.Specific item is not specified in embodiment
The experimental method of part, usually according to normal condition.In the embodiment of the present invention unless otherwise noted, agents useful for same and consumptive material are city
Sell commodity.
Embodiment 1
1, for detecting the primer and blocking agent of human EGFR gene T790M mutation
It is reference with the mutational site EGFR according to the EGFR gene wild-type sequence that ncbi database is announced, design is special
Property EGFR mutant primer.Using the mutant plasmid of genetic engineering building and wild plasmid as template, real-time fluorescence PCR is established
It is mutated (Mutation) detection architecture, realizes and the human EGFR gene T790M high sensitivity being mutated and high specific is detected.
For detecting the primer sequence of human EGFR gene T790M mutation:
Mutational site EGFR (c.2369C > T)
It is mutated upstream primer:
5’-TCTGCCTCACCTCCACCGTGCAGCTCATCATGC-3’(SEQ ID NO.1)
It is mutated downstream primer:
5’-AATATTGTCTTTGTGTTCCCGGAC-3’(SEQ ID NO.2)。
Wherein, upstream primer, in conjunction with EGFR (c.2369C > T) site mutation sequence-specific, selective amplification are mutated
Mutant nucleotide sequence；It is mutated downstream primer, in conjunction with EGFR gene conserved sequence.
In order to further discriminate between wild-type sequence and mutant sequences, the present invention is added to blocking agent, including matching EGFR
The nucleic acid sequence and chemical modification group of wild type gene；
Wherein, the nucleic acid sequence of EGFR wild type gene is matched in the blocking agent are as follows:
5'-AGCTCATCACGCAGCTCAT-3'(SEQ ID NO.3)；
The chemical modification group is C3-spacer phosphamide.
Above-mentioned blocking agent can be specifically bound with EGFR gene T790M site wild-type sequence, inhibit wild type gene non-
Specific amplified.
2, the detection kit of human EGFR gene T790M mutation provided by the invention, operating procedure are as follows:
1) blood DNA sample is extracted, spare:
2) dosage of component prepares qRT-PCR reaction system according to the form below:
| Reagent | Dosage |
| SYBR MIX | 10μl |
| It is mutated upstream primer (4 μM) | 1.0μl |
| It is mutated downstream primer (4 μM) | 1.0μl |
| ddH2O | 4.0μl |
| Blocking agent (1 μM) | 2.0μl |
| DNA | 2.0μl |
3) qRT-PCR reacts:
QRT-PCR reaction condition: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 60 DEG C of extension 1min (collecting fluorescence), into
Row 40 circulations.
3, the performance verification of kit
1) preparation of detection template:
Take the genetic engineering bacterium containing EGFR gene T790M mutant plasmid and corresponding wild plasmid genetic engineering bacterium
Cultivate respectively, use the plasmid of plasmid purification kit (Plasmid Mini Kit be purchased from OMEGA company) preparation purifying as
Detection template.
2) effect of blocking agent:
Since the target gene segment EGFR gene T790M of this reagent detection is mutated the point mutation of only one base, this
The detection primer that invention provides is in the system that blocking agent is not added when detecting wild type gene it is possible that false positive, such as schemes
In 2 (abscissa is the CT value of qPCR, and ordinate is detected fluorescent increment) shown in amplification curve B1, and blocked being added
Due to the presence of blocking agent in the system of agent, it is suppressed that the amplification of wild type gene, as Fig. 2 amplification curve B2 (does not expand) institute
Show, the results showed that blocking agent, which is added, can inhibit the amplification of wild type gene；And the system of blocking agent is added and blocking agent is not added
System, the two is to the sensitive similar temperament of detection mutated genes, its CT value of amplification curve A1 (blocking agent is not added) is 20, and amplification is bent
The CT value of line A2 (adding blocking agent) is 21, the results showed that when amplification mutated gene, blocking agent is added to only have a CT to mutated gene
Delay, adding and blocking agent is not added influences very little to the amplification of mutated gene, but adds blocking agent that can obviously inhibit wild type base
The amplification of cause greatly reduces the interference that a large amount of wild type genes detect mutated genes, the more conducively detection of mutated genes.
3) sensitivity of kit:
It prepares the plasmid of purifying and carries out nucleic acid quantification, calculate copy number, 10 times of gradient dilutions take 108Copy/μ L,
107Copy/μ L, 106Copy/μ L, 105Copy/μ L mutant plasmids are added 1010It is dashed forward in copy/μ L wild plasmid
Modification gene accounts for the sample that the ratio of wild type gene is 1%, 0.1%, 0.01%, 0.001%, takes 2 each dilutions of μ L respectively
Plasmid DNA as template, the sensitivity of kit is detected, as a result as shown in figure 3, provided by the present invention for inspection
The kit for surveying human EGFR gene T790M mutation is equal when mutated gene concentration is 1%, 0.1%, 0.01%, 0.001%
It can effectively expand, detection sensitivity can reach 0.001%.
4) specificity of kit:
Respectively using Sudden Anomalies gene, normal gene, the similar non-targeted gene of mutation of other relatively conventional sequences as
Detection template is detected, with the specificity of detection kit.As a result as shown in figure 4, wherein A is to expand Sudden Anomalies gene
Increase, B be to normal gene amplification, C be to TP53 gene magnification, D be KIT gene magnification, E is to non-targeted gene compound
Amplification.The result shows that can specific detection EGFR provided by the present invention for the kit of detection human EGFR gene T790M mutation
Gene T790M (c.2369C > T) site mutation.
In conclusion the kit of detection human EGFR gene T790M mutation provided by the invention can detect EGFR gene
T790M (c.2369C > T) site mutation, and kit sensitivity with higher and specificity are detection human EGFR genes
A kind of ideal chose of T790M mutation.
The above embodiments are merely illustrative of the technical solutions of the present invention rather than limiting the scope of the invention, although ginseng
The present invention is explained in detail according to preferred embodiment, those skilled in the art should understand that, it can be to of the invention
Technical solution is modified or replaced equivalently, without departing from the spirit and scope of technical solution of the present invention.
Sequence table
<110>Guangzhou Jian Tian gene Technology Co., Ltd.
<120>for detecting primer, detection method and the kit of human EGFR gene T790M mutation
<150> 2017109552044
<151> 2017-10-13
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tctgcctcac ctccaccgtg cagctcatca tgc 33
<210> 2
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
aatattgtct ttgtgttccc ggac 24
<210> 3
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
agctcatcac gcagctcat 19
Claims (10)
1. the detection method for detecting human EGFR gene T790M mutation, comprising the following steps: will be used to detect human EGFR
The primer of gene T790M mutation, blocking agent, detection fluorescence signal group, the mixing of testing gene sample, carry out real time fluorescent quantitative
PCR obtains testing result；
Wherein, the primer being mutated for detecting human EGFR gene T790M, including mutation upstream primer and mutation downstream are drawn
Object, mutation upstream primer include the primer that 1-10 item is directed to different mutational sites, and the length is 15-40 bases, close to 3 ' ends
1st, 2,3,4 or 5 base at end is mutational site, complementary with a chain of EGFR mutated genes to be measured；It is mutated downstream
Primer is a shared downstream primer, and the length is 15-40 bases, mutual with another chain of EGFR mutated genes to be measured
It mends；
The blocking agent can inhibit the non-specific expansion of wild type with the mutational site EGFR gene T790M wild-type sequence specific bond
Increase；
The detection fluorescence signal group in conjunction with pcr amplified fragment and can issue fluorescence signal.
2. as described in claim 1 for detecting the detection method of human EGFR gene T790M mutation, which is characterized in that packet
Include 1 primer for being directed to mutational site, the length is 33 bases, wherein the 3rd bit base of 3 ' ends is mutational site, with to
Survey a chain complementation of EGFR mutated genes.
3. as described in claim 1 for detecting the detection method of human EGFR gene T790M mutation, the mutation downstream is drawn
Object, the length is 24 bases, complementary with another chain of EGFR mutated genes to be measured.
4. as described in claim 1 for detecting the detection method of human EGFR gene T790M mutation, which is characterized in that institute
Stating primer sequence includes:
Mutational site EGFR (c.2369C > T)
It is mutated upstream primer:
TCTGCCTCACCTCCACCGTGCAGCTCATCATGC(SEQ ID NO.1)
It is mutated downstream primer:
AATATTGTCTTTGTGTTCCCGGAC(SEQ ID NO.2)。
5. as described in claim 1 for detecting the detection method of human EGFR gene T790M mutation, which is characterized in that institute
Stating blocking agent includes: the nucleic acid sequence for matching EGFR wild type gene, and the nucleic acid sequence is modified with chemical group；
Wherein, the nucleic acid sequence for EGFR wild type gene being matched in the blocking agent includes: AGCTCATCACGCAGCTCAT (SEQ
ID NO.3)；The chemical group includes: one of amido, phosphate group, biotin, biotin-TEG and C3-spacer
Or it is a variety of.
6. as described in claim 1 for detecting the detection method of human EGFR gene T790M mutation, which is characterized in that institute
Stating detection fluorescence signal group includes: FAM, Cy3, HEX, Texas Red, JOE, VIC, TAMRA, Cy5, SYTO9, SYBR MIX
One of or it is a variety of.
7. the primer for detecting human EGFR gene T790M mutation, which is characterized in that including under mutation upstream primer and mutation
Primer is swum, mutation upstream primer includes the primer that 1-10 item is directed to different mutational sites, close the length is 15-40 base
1st, 2,3,4 or 5 base of 3 ' ends is mutational site, complementary with a chain of EGFR mutated genes to be measured；Mutation
Downstream primer is a shared downstream primer, and the length is 15-40 bases, another with EGFR mutated genes to be measured
Chain is complementary.
8. the blocking agent for detecting human EGFR gene T790M mutation, which is characterized in that the blocking agent energy and human EGFR
The mutational site gene T790M wild-type sequence specific bond inhibits wild type non-specific amplification.
9. the kit for detecting human EGFR gene T790M mutation, which is characterized in that including as claimed in claim 7
For detecting the primer of human EGFR gene T790M mutation, as claimed in claim 8 for detecting human EGFR gene
The blocking agent of T790M mutation further includes detection fluorescence signal group.
10. described in the detection method as described in claim 1 for being used to detect human EGFR gene T790M mutation, claim 7
Primer for detecting human EGFR gene T790M mutation and/or according to any one of claims 8 for detecting human EGFR gene
T790M mutation kit detection human EGFR gene mutations in application, it is described apply it is not low in mutated genes concentration
Application in 1%, 0.1%, 0.01% or 0.001% sample detection human EGFR gene mutations.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710955204 | 2017-10-13 | ||
| CN2017109552044 | 2017-10-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109306379A true CN109306379A (en) | 2019-02-05 |
Family
ID=65224911
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811044900.0A Pending CN109306379A (en) | 2017-10-13 | 2018-09-07 | For detecting primer, detection method and the kit of human EGFR gene T790M mutation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109306379A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110004219A (en) * | 2019-04-19 | 2019-07-12 | 格尚微(上海)生物科技有限公司 | A kind of detection method of the cis- mutation of EGFR T790M and C797S |
| CN110373467A (en) * | 2019-07-29 | 2019-10-25 | 温州医科大学 | A kind of detection reagent and detection method of human EGFR gene mutations site T790M |
| CN110438226A (en) * | 2019-07-25 | 2019-11-12 | 深圳市优圣康生物科技有限公司 | A kind of kit, sample treatment and the determination method of the mutation of detection cis-trans |
| CN112481366A (en) * | 2020-12-29 | 2021-03-12 | 苏州科诺医学检验实验室有限公司 | Primer pair and kit for detecting EGFR gene T790M mutation in plasma free DNA |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105238869A (en) * | 2015-11-11 | 2016-01-13 | 北京市农林科学院 | FLC1 specific SNP molecular marker based on competitive allele specificity PCR technology and application |
| WO2016172265A1 (en) * | 2015-04-20 | 2016-10-27 | Neogenomics Laboratories, Inc. | Method to increase sensitivity of next generation sequencing |
| CN107012221A (en) * | 2016-10-14 | 2017-08-04 | 苏州艾达康医疗科技有限公司 | Enrichment system and its application based on blocker |
-
2018
- 2018-09-07 CN CN201811044900.0A patent/CN109306379A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016172265A1 (en) * | 2015-04-20 | 2016-10-27 | Neogenomics Laboratories, Inc. | Method to increase sensitivity of next generation sequencing |
| CN105238869A (en) * | 2015-11-11 | 2016-01-13 | 北京市农林科学院 | FLC1 specific SNP molecular marker based on competitive allele specificity PCR technology and application |
| CN107012221A (en) * | 2016-10-14 | 2017-08-04 | 苏州艾达康医疗科技有限公司 | Enrichment system and its application based on blocker |
Non-Patent Citations (3)
| Title |
|---|
| CRISTIN ROMA ET AL: "Detection of EGFR Mutations by TaqMan Mutation Detection Assays Powered by Competitive Allele-Specific TaqMan PCR Technology", 《BIOMED RESEARCH INTERNATIONAL》 * |
| LUMING ZHOU等: "Rare allele enrichment and detection by allele-specific PCR, competitive probe blocking, and melting analysis", 《BIOTECHNIQUES》 * |
| 李金春等: "引物3’端不同碱基错配情况下实时荧光定量PCR非特异性扩增的发生规律", 《食品科学》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110004219A (en) * | 2019-04-19 | 2019-07-12 | 格尚微(上海)生物科技有限公司 | A kind of detection method of the cis- mutation of EGFR T790M and C797S |
| CN110438226A (en) * | 2019-07-25 | 2019-11-12 | 深圳市优圣康生物科技有限公司 | A kind of kit, sample treatment and the determination method of the mutation of detection cis-trans |
| CN110438226B (en) * | 2019-07-25 | 2021-09-03 | 深圳市优圣康生物科技有限公司 | Kit for detecting cis-trans mutation, sample processing method and judgment method |
| CN110373467A (en) * | 2019-07-29 | 2019-10-25 | 温州医科大学 | A kind of detection reagent and detection method of human EGFR gene mutations site T790M |
| CN112481366A (en) * | 2020-12-29 | 2021-03-12 | 苏州科诺医学检验实验室有限公司 | Primer pair and kit for detecting EGFR gene T790M mutation in plasma free DNA |
| CN112481366B (en) * | 2020-12-29 | 2022-08-16 | 苏州科诺医学检验实验室有限公司 | Primer pair and kit for detecting EGFR gene T790M mutation in plasma free DNA |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Harms et al. | Distinct gene expression profiles of viral-and nonviral-associated merkel cell carcinoma revealed by transcriptome analysis | |
| CN109306379A (en) | For detecting primer, detection method and the kit of human EGFR gene T790M mutation | |
| JP7256840B2 (en) | Splice variants associated with neomorphic SF3B1 mutants | |
| Pressey et al. | Hedgehog pathway activity in pediatric embryonal rhabdomyosarcoma and undifferentiated sarcoma: a report from the Children's Oncology Group | |
| US20110269640A1 (en) | PROBES FOR DETECTING MUTATIONS OF kRas GENE, LIQUICHIP AND DETECTION METHODS THEREOF | |
| CN103710460B (en) | Test kit of detection by quantitative EGFR genetic mutation and uses thereof | |
| WO2012157684A1 (en) | Method for assessing progression of clinical state of malignant neoplasm by quantitative detection of dna in blood | |
| CN104350161A (en) | Compositions and methods for sensitive mutation detection in nucleic acid molecules | |
| JP2010510769A (en) | Methods and compositions for diagnosis of esophageal cancer and prognosis and improvement of patient survival | |
| WO2015180489A1 (en) | Nras gene mutation detection kit | |
| CN112522408A (en) | Methods and compositions relating to DNA damaging agents for cancer treatment | |
| EP3704274B1 (en) | Genetic regulation of immunoresponse by chromosome interactions | |
| US20200354794A1 (en) | Method for determining sensitivity to simultaneous inhibitor against parp and tankyrase | |
| CN107164513A (en) | Mankind's polygenic mutation detection kit based on high-flux sequence method | |
| CN112824535A (en) | Primer composition for gene mutation multiplex detection and kit thereof | |
| CN109295177A (en) | A kind of micro mutation of human EGFR gene is enriched with pretreated primer, method and kit | |
| CN102796811B (en) | Reagent and method for detecting KRAS mutation | |
| Wang et al. | Universal and highly accurate detection of circulating tumor DNA mutation in non-small cell lung cancer based on CRISPR/Cas12a system | |
| CN109306374A (en) | For detecting primer, detection method and the kit of mankind PIK3CA gene E545K mutation | |
| CN108130362A (en) | Kit and application for EGFR genetic mutation detection | |
| Gamberi et al. | Detection of H3F3A p. G35W and p. G35R in giant cell tumor of bone by Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR) | |
| CN107354197B (en) | Kit for detecting human NRAS gene mutation | |
| CN103930567B (en) | NEW TYPE OF COMPOSITE sudden change in epidermal growth factor receptor kinase domain | |
| Chen et al. | Distribution and characterization of extrachromosomal circular DNA in colorectal cancer | |
| CN109295176A (en) | For detecting primer, detection method and the kit of Human Colorectal Cancer BRAF gene V600E 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 | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20190213 Address after: 510663 Room A701, Guangzhou International Business Incubator Area A, No. 3 Zhuquan Road, Science City, Guangzhou High-tech Industrial Development Zone, Guangdong Province Applicant after: Guangzhou Jiantian Pharmaceutical Technology Co.,Ltd. Address before: 510663 Room 701, Guangzhou International Business Incubator Area A, No. 3 Shenquan Road, Science City, Guangzhou High-tech Industrial Development Zone, Guangdong Province Applicant before: GUANGZHOU GENETX TECHNOLOGY CO.,LTD. |
|
| TA01 | Transfer of patent application right | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190205 |
|
| RJ01 | Rejection of invention patent application after publication |