CN109457018A - A kind of EGFR mutated gene detection method based on multiple fluorescence PCR - Google Patents
A kind of EGFR mutated gene detection method based on multiple fluorescence PCR Download PDFInfo
- Publication number
- CN109457018A CN109457018A CN201811518772.9A CN201811518772A CN109457018A CN 109457018 A CN109457018 A CN 109457018A CN 201811518772 A CN201811518772 A CN 201811518772A CN 109457018 A CN109457018 A CN 109457018A
- Authority
- CN
- China
- Prior art keywords
- seq
- egfr
- mutated gene
- sequence
- detection
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The EGFR mutated gene detection method based on multi-fluorescence that the invention discloses a kind of, is related to the detection field of EGFR mutated gene.The EGFR mutated gene detection method based on multiple fluorescence PCR that the invention proposes a kind of, this method passes through the blocker for preparing a variety of specific ARMS- amplimers for different EGFR mutated genes and wild type gene capable of being inhibited to expand, the segment containing mutated gene in measuring samples is expanded, is judged in different samples according to fluorescent value with the presence or absence of corresponding EGFR genetic mutation.The present invention proposes a kind of kit using this method detection EGFR mutated gene simultaneously.The advantages of present invention has high sensitivity, accuracy height, high specificity, can detect a variety of EGFR mutated genes simultaneously.
Description
Technical field
The present invention relates to cancer patient EGFR mutated gene detection fields more particularly to a kind of based on multiple fluorescence PCR
EGFR mutated gene detection method.
Background technique
Human epidermal growth factor (epidermal growth factor receptor, EGFR) is a kind of albumen junket ammonia
Acid kinase receptor is distributed widely in the cell surfaces such as mammalian epithelial cell, fibroblast, spongiocyte, has junket ammonia
Kinase activity.EGFR is one of HER/ErbB family member.EGFR forms dimer in cell surface with after its ligand binding,
By the activity of tyrosine kinase, activated receptor autophosphorylation is adjusted by the cascade reaction of adaptin and enzyme in cytoplasm
The transcription for saving transcription factor activation gene, instructs cell migration, proliferation, differentiation and apoptosis.When EGFR mutates, EGFR is certainly
Body or ligand are overexpressed, to stimulate cell to form uncontrolled proliferation by autocrine or paracrine mode.In addition, EGFR is excessive
Activation can also start the expression of multiple protein hydrolysis and angiogenic factors, to accelerate the transfer of cancer cell.Therefore,
The usual prognosis of EGFR overexpression person is poor.
Iressa and Erlotinib are that U.S. FDA approval is targeted for NSCLC as EGFR receptor tyrosine kinase inhibitors
The key agents for the treatment of.But clinical trial shows that Iressa and Erlotinib only have significant treatment to patient NSCLC of 10-30%
Effect.Further investigation revealed that EGFR genetic mutation is related to NSCLC targeted therapy curative effect, majority carries EGFR genetic mutation
Patient is significant in efficacy.There is the patient of EGFR tyrosine kinase gene code area 18-21 exons mutation in lung carcinoma cell, targets medicine
The effective percentage of object Gefitinib is up to 80%.If patient does not carry EGFR genetic mutation and take such drug, not only can
Delay the state of an illness also and will cause the waste of huge medication resource.Therefore, it detects whether prominent containing EGFR gene in tissue or blood
Become for instructing NSCLC patients clinical medication, the development side with important reference value and future tumors individualized treatment
To.
EGFR sports somatic mutation, and the frequency being mutated in tumor patient is higher (about 10% in U.S. NSCLC patient
There is an EGFR genetic mutation, and 30%) asian population this ratio is about.But mutated gene and the wild type base not mutated
Because comparing, mutant proportion is extremely low, and according to different neoplasm stagings, this ratio is usually 0.01%~10%.Therefore it is directed to EGFR
The maximum problem of detection of mutated gene is how under a large amount of wild type gene backgrounds to detect that minute quantity mutates
EGFR gene.In addition, due to known more than 40 kinds can be up to the related EGFR mutation type of targeted therapy, how to realize
Effect 40 various mutations types of detection are another challenges.The method of detection gene mutation at present specifically includes that direct sequencing, passes
The fluorescence quantitative PCR method and probe amplification of system block mutation method (amplification refractory mutation
System, ARMS) (application number CN101608240B/CN102747157A).Time-consuming and sensitivity is low for direct sequencing, can only
The gene mutation for being more than 5% to content detects, and can typically be only used to the scientific research in laboratory, 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
There is the testing result of false positive；The flux of the bit number of points of detection and detection sample is all relatively small simultaneously.ARMS-PCR
Method is the principle that could must be effectively expanded with template complete complementary using 3 ' terminal bases of PCR primer, is directed to by design
The specific PCR amplimer in mutational site, and then achieve the purpose that detect mutated gene.Although ARMS-PCR method is a kind of
Relatively simple and with higher sensitivity detection method, but it is constrained to PCR sense channel, it is difficult to it realizes and a variety of EGFR is mutated
Gene is carried out while being detected.It following is a brief introduction of existing method and its defect.
Direct sequencing: first designing corresponding primer for mutational site, then obtains target gene by PCR amplification and produces
Object is finally sequenced PCR product, and analyzes sequencing result.The sensitivity of PCR sequencing PCR is low, can only be more than to content
5% gene mutation is detected.Therefore the method is not suitable for the analysis for being clinically applied to a large amount of clinical samples.
Denaturing high-performance liquid chromatography: the principle of this method is based on the pure of the heteroduplex and exact matching that mispairing occurs
The difference for closing double-stranded DNA unwinding feature separates the two.Since there is mispairing in mutational site in heteroduplex, it is easier to be formed
Special structure is reduced with the binding ability of solid phase, therefore heterozygosis chain is preferentially eluted out than homozygous chain, passes through eluting peak
Difference determines whether the presence of mutation.But this method expensive equipment, to the professional proficient of instrument and equipment and operator
There is very high requirement.Simultaneously when detecting, relatively high to the purity requirement of sample, there is also under-sensitive problem (loadings
Amount is greater than 0.01ng).
High-resolution solubility curve (application number CN104762408B): the physical property based on nucleic acid passes through saturable dye
It is incorporated into pcr amplification product, monitors the mutation analysis gene mutation of product solubility curve.Detection sensitivity 5% or so, for
Positive findings not can determine that its specific location, finally need to be confirmed by PCR sequencing PCR.
Probe amplification blocks the (Shen mutation method (amplification refractory mutation system, ARMS)
Please number CN101608240B/CN102747157A): complementary with template could must effectively it be expanded using 3 terminal bases of PCR primer
The principle of increasing, design are directed to the specific PCR amplimer in mutational site, and then achieve the purpose that detection mutation.Although ARMS-
PCR method is a kind of relatively simple and with higher sensitivity detection method, but is constrained to PCR sense channel, it is difficult to be realized
The primary of EGFR mutated gene detects simultaneously.
Therefore, low with detection flux in order to solve the sensitivity that existing EGFR mutated gene detection technique is faced simultaneously
Problem, this invention address that developing a kind of new mutational site EGFR detection method and kit.This method should have sensitive
The advantages of spending high, accuracy height and high specificity, and capable of detecting various mutations gene simultaneously.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of detection sides that can detect a variety of mutational sites EGFR simultaneously
Method, this method can guarantee the accuracy and detection flux of sensitivity and the detection of detection method simultaneously, while this method is also easy
In large-scale promotion.
To achieve the above object, the EGFR mutated gene detection method based on multiple fluorescence PCR that the present invention provides a kind of,
Characterized by comprising the following steps:
Step 1: preparation is directed to the detection probe in the different mutational sites EGFR, the detection probe respectively corresponds one
The mutational site EGFR, detection probe described in each includes the sequence of 15-25 base, in the medium position of the sequence, institute
The both ends for stating detection probe are marked with fluorescence detection group and quenching group respectively；
Step 2: the specific ARMS amplimer in the corresponding different mutational sites EGFR of preparation, anti-using multiple fluorescence PCR
Answer the segment containing mutated gene in system amplification measuring samples；
Step 3: preparation corresponds to the blocker in the different mutational sites EGFR to inhibit the amplification of wild-type genotype, recycle
Multiple fluorescence PCR reaction system expands the segment containing mutated gene in measuring samples；
Step 4: determining measuring samples with the presence or absence of corresponding EGFR mutation type according to fluorescence signal.
Further, the detection probe is at least one sequence or its reverse complemental in SEQ ID NO.1-ID 7
The DNA of sequence.
Further, the fluorescence detection group is preferably FAM, ROX, CY5 and HEX one or more.
In a preferred embodiment of the present invention, the blocker sequential covering mutational site sequence in the third step,
It is matched completely with wild type sequence, at least there is a base mispairing with mutant nucleotide sequence, the sequence length of the blocker is
10bp-25bp is modified containing PNA or LNA in the blocker series.
Further, the detection probe is at least one sequence or its reverse complemental in SEQ ID NO.1-ID 7
The DNA of sequence.
The multiple fluorescence PCR detection architecture being mutated for diagnosing EGFR, including the label detection of different fluorophors are visited
Needle (table 1), probe described in each respectively correspond the mutational site EGFR, and the mutational site EGFR is G719X, 19-
At least one of del, 20-insertions, T790M, S768I, L858R and L861Q；Detection probe described in each includes
The sequence of 15-20 base includes the amplification base sequence of its corresponding EGFR mutation in the medium position of the sequence.
Further, each described detection probe corresponds to the EGFR mutation type described at least one, wherein SEQ
The corresponding mutation type of ID NO.1 is EX19DEL, and the corresponding mutation type of SEQ ID NO.2 is G719X, SEQ ID NO.3 couple
The mutation type answered is EX20ins, and the corresponding mutation type of SEQ ID NO.4 is L858R, the corresponding mutation of SEQ ID NO.5
Type is T790M, and the corresponding mutation type of SEQ ID NO.6 is S768I, and the corresponding mutation type of SEQ ID NO.7 is
L861Q, SEQ ID NO.36 corresponding is internal control Sequence Detection.
In a preferred embodiment of the invention, the PCR primer for being used to expand sample to be examined genetic fragment
(see Table 2 for details), the amplified production of the PCR primer contain above-mentioned gene mutation site, and the PCR primer is following 17 pairs
In it is at least a pair of: SEQ ID NO.8 and SEQ ID NO.9；SEQ ID NO.10 and SEQ ID NO.11；SEQ ID NO.10
With SEQ ID NO.12；SEQ ID NO.10 and SEQ ID NO.13；SEQ ID NO.10 and SEQ ID NO.14；SEQ ID
NO.15 and SEQ ID NO.16；SEQ ID NO.15 and SEQ ID NO.17；SEQ ID NO.15 and SEQ ID NO.18；SEQ
ID NO.15 and SEQ ID NO.19；SEQ ID NO.15 and SEQ ID NO.20；SEQ ID NO.15 and SEQ ID NO.21；
SEQ ID NO.22 and SEQ ID NO.23；SEQ ID NO.24 and SEQ ID NO.25；SEQ ID NO.26 and SEQ ID
NO.27；SEQ ID NO.28 and SEQ ID NO.29；The amplified production of SEQ ID NO.8 and SEQ ID NO.9 includes
EX19DEL mutation type；SEQ ID NO.10 and SEQ ID NO.11/SEQ ID NO.12/SEQ ID NO.13/SEQ ID
The amplified production of NO.14 includes G719X mutation type；SEQ ID NO.15 and SEQ ID NO.16/SEQ ID NO.17/SEQ
The amplified production of ID NO.18/SEQ ID NO.19/SEQ ID NO.20/SEQ ID NO.21 includes EX20ins mutation type；
The amplified production of SEQ ID NO.22 and SEQ ID NO.23 includes L858R mutation type；SEQ ID NO.24 and SEQ ID
The amplified production of NO.25 includes T790M mutation type；The amplified production of SEQ ID NO.26 and SEQ ID NO.27 includes
S768I mutation type；The amplified production of SEQ ID NO.28 and SEQ ID NO.29 includes L861Q mutation type；SEQ ID
The amplified production of NO.37 and SEQ ID NO.38 includes internal control sequence.
Preferably, the PCR primer can combine in different ways to carry out simultaneously different loci in single reaction pipe
Amplification: SEQ ID NO.8/SEQ ID NO.9/SEQ ID NO.15/SEQ ID NO.16/SEQ ID NO.17/SEQ ID
NO.18/SEQ ID NO.19/SEQ ID NO.20/SEQ ID NO.21 can be expanded simultaneously comprising EX19DEL's and EX20ins
Mutation type；SEQ ID NO.22/SEQ ID NO.23/SEQ ID NO.24/SEQ ID NO.25 can be expanded simultaneously includes
The mutation type of L858R and T790M；SEQ ID NO.10/SEQ ID NO.11/SEQ ID NO.12/SEQ ID NO.13/
SEQ ID NO.14/SEQ ID NO.26/SEQ ID NO.27/SEQ ID NO.28 and SEQ ID NO.29 can expand packet simultaneously
Mutation type containing G719X/S768I/L861Q；
Further, the blocker sequence is at least one of SEQ ID NO.30-SEQID 35 (see Table 3 for details)
Sequence or its reverse complemental DNA sequence dna, each described blocker correspond to the EGFR mutated gene class described at least one
Type, wherein the corresponding mutation type of SEQ ID NO.30 is 19DEL, and the corresponding mutation type of SEQ ID NO.31 is G719D/
The corresponding mutation type of G719A/G719S/G719C, SEQ ID NO.32 is L858R, the corresponding mutation class of SEQ ID NO.33
Type is T790M, and the corresponding mutation type of SEQ ID NO.34 is S768I, and the corresponding mutation type of SEQ ID NO.35 is
L861Q。
The present invention also provides a kind of kits for detecting EGFR mutated gene, it is characterised in that the detection EGFR mutation
The kit of gene is utilized the above-mentioned EGFR mutated gene detection method based on multiple fluorescence PCR and is detected.Including more
Weight fluorescent PCR detection architecture, and contain the above-mentioned PCR primer for being used to expand measuring samples genetic fragment and blocker
PCR reaction solution.
Table 1: probe sequence used in abrupt climatic change
Primer sequence needed for table 2:ARMS-PCR amplification
Blocker sequence needed for table 3:ARMS-PCR amplification
The present invention is compared with the prior art beneficial effect: the present invention be amplification technique based on ARMS and Blocker and
Detection in Gene Mutation technology based on specific probe.Since the common presence of ARMS and Blocker can be directed to required detection
Mutation type expanded, then amplified production is detected by specific probe.It in this way can be directly to person's to be checked
Genotype is made a definite diagnosis, and can play good judgement to the type of mutation, has that accuracy is high, high specificity, can detect compared with
Low mutated gene copy number and ratio.
It is described further below with reference to technical effect of the attached drawing to design of the invention, specific structure and generation, with
It is fully understood from the purpose of the present invention, feature and effect.
Detailed description of the invention
Fig. 1 is the testing result of EGFR gene hot spot mutation 19DEL/20INSERT；
Fig. 2 is the testing result of EGFR gene hot spot mutation T790M/L858R；
Fig. 3 is the testing result of EGFR gene hot spot mutation G719X/S768I/L861Q.
Specific embodiment
Multiple preferred embodiments of the invention are introduced below with reference to Figure of description, keep its technology contents more clear and just
In understanding.The present invention can be emerged from by many various forms of embodiments, and protection scope of the present invention not only limits
The embodiment that Yu Wenzhong is mentioned.
Technology contents of the invention are described further below with reference to embodiment: following embodiments be it is illustrative,
It is not restrictive, cannot be limited the scope of protection of the present invention with following embodiments.Test as used in the following examples
Method is conventional method unless otherwise specified.The materials, reagents and the like used in the following examples, unless otherwise specified,
It obtains from commercial channels.
Embodiment one: (the E746-A750del/EX20ins detection on EGFR gene hot spot mutation exons 1 9)
1, template DNA is extracted:
Extracting template DNA from the tissue samples of patient using other commercial kits, (including flesh tissue, paraffin are cut
Piece, pleural effusion and whole blood).In addition, by be separately added into reaction solution 10000 copy wild template (A549) and
The mutated genes (Del 19/20insert) of different proportion, then carrying out augmentation detection through the invention again can to verify it
By property.
2, PCR amplification
According to the positional relationship of this project EGFR mutation type to be checked, we devise 7 pairs of primers for EX19DEL/
EX20ins different mutation types are expanded, and the amplified production of SEQ ID NO.8 and SEQ ID NO.9 is prominent comprising EX19DEL
Become type；SEQ ID NO.15 and SEQ ID NO.16/SEQ ID NO.17/SEQ ID NO.18/SEQ ID NO.19/SEQ
The amplified production of ID NO.20/SEQ ID NO.21 includes EX20ins mutation type.
2.1, synthetic pcr primer object/specific probe: synthetic method is existing conventional DNA synthetic method.
2.2, prepare PCR reaction solution: preparing the PCR reaction solution of 15 microlitres/person-portion, in everyone PCR reaction solution divided respectively at
Point and concentration be respectively as follows: upstream primer concentration be respectively 0.2 it is micro- rub/liter, Taq enzyme is 1U/ microlitres micro-, 1X PCR buffer,
MgCl2 be 1.5 mmoles/liter, dNTP be 0.2 mmoles/liter, template DNA 1-10 nanogram/microlitre.
2.3, PCR amplification: the response procedures of PCR amplification are as follows: 95 DEG C initial denaturation 5 minutes；95 DEG C are denaturalized 30 seconds, 70 DEG C 20
Second, 60 DEG C renaturation 30 seconds, 72 DEG C extend 30 seconds, recycle 35 times.Product after PCR amplification includes the DNA fragmentation of patient to be detected.
3, signal collection
Corresponding fluorescence signal is collected at 60 DEG C of the second stage in PCR amplification program.The present invention utilizes modified-
ARMS-PCR technology distinguishes wild type and mutated genes sequence, judges by the fluorescence intensity in reaction system detection knot
Fruit.VIC is internal standard signal, for detecting whether leakage plus template.
Outer mark system is used for the quality of detection template, and external control system is individually, so not influenced by double reaction, outside
The C of table system signaltValue should be less than 30, and it is too low to be otherwise considered as template concentrations, and testing requirements are not achieved.
Detection architecture is for detecting whether there are mutational sites: after internal standard and external standard signal reach requirement, to detect body
Cycle-index C needed for fluorescence signal in system reaches given thresholdtValue is used as judgment criteria.
Fig. 1 is the testing result of EGFR gene hot spot mutation 19DEL/20INSERT, and 1. is wild for the A549 of 10000 copies
Under type genome background, the ID39 plasmid (channel CY5) of 100 copies；2. for the A549 wild type gene group background of 10000 copies
Under, the plasmid (channel FAM) of the ID42 of 100 copies；3. for the A549 wild type gene group (channel VIC) of 10000 copies；4. being
The A549 wild type gene group CY5 Air conduct measurement of 10000 copies；5. for the channel A549 wild type gene group FAM of 10000 copies
Detection.
Embodiment two: the abrupt climatic change of EGFR gene hot spot mutation exon T790M/L858R
1, template DNA is extracted:
Extracting template DNA from the tissue samples of patient using other commercial kits, (including flesh tissue, paraffin are cut
Piece, pleural effusion and whole blood).In addition, by be separately added into reaction solution 10000 copy wild template (A549) and
Then the mutated genes (T790M/L858R) of different proportion carry out augmentation detection through the invention again to verify its sensitivity.
2, PCR amplification
According to the positional relationship of this project EGFR mutation type to be checked, we devise 2 pairs of primers for L858R/
T790M different mutation types are expanded, and the amplified production of SEQ ID NO.22 and SEQ ID NO.23 is mutated comprising L858R
Type；The amplified production of SEQ ID NO.24 and SEQ ID NO.25 includes T790M mutation type.
Synthetic pcr primer object/specific probe: synthetic method is existing conventional DNA synthetic method.
Prepare PCR reaction solution: preparing the PCR reaction solution of 15 microlitres/person-portion, in the PCR reaction solution of every person-portion each ingredient and
Concentration be respectively as follows: upstream and downstream primer and blocker sequence concentration be respectively 0.2 it is micro- rub/liter, Taq enzyme is 1U/ microlitres micro-, 1X
PCR buffer, MgCl2 be 1.5 mmoles/liter, dNTP be 0.2 mmoles/liter, template DNA 1-10 nanogram/microlitre.
PCR amplification: the response procedures of PCR amplification are as follows: 95 DEG C initial denaturation 5 minutes；95 DEG C be denaturalized 30 seconds, 70 DEG C 20 seconds, 60
DEG C renaturation 30 seconds, 72 DEG C extended 30 seconds, recycled 35 times.Product after PCR amplification includes the DNA fragmentation of patient to be detected.
3, signal collection
Corresponding fluorescence signal is collected at 60 DEG C of the second stage in PCR amplification program.The present invention utilizes modified-
ARMS-PCR technology distinguishes wild type and mutated genes sequence, judges by the fluorescence intensity in reaction system detection knot
Fruit.VIC is internal standard signal, for detecting whether leakage plus template.
Outer mark system is used for the quality of detection template, and external control system is individually, so not influenced by double reaction, outside
The CT value of table system signal should be less than 30, and it is too low to be otherwise considered as template concentrations, and testing requirements are not achieved.
Detection architecture is for detecting whether there are mutational sites: after internal standard and external standard signal reach requirement, to detect body
Cycle-index Ct value is as judgment criteria needed for fluorescence signal in system reaches given threshold.
Fig. 2 is the testing result of EGFR gene hot spot mutation T790M/L858R, the 1. A549 wild types copied for 10000
Under genome background, the ID39 plasmid (channel CY5) of 100 copies；2. for the A549 wild type gene group background of 10000 copies
Under, the L858R plasmid (channel FAM) of 100 copies；3. for the A549 wild type gene group (channel VIC) of 10000 copies；4. being
The A549 wild type gene group CY5 Air conduct measurement of 10000 copies；5. for the channel A549 wild type gene group FAM of 10000 copies
Detection.
Embodiment three:
1, template DNA is extracted:
Extracting template DNA from the tissue samples of patient using other commercial kits, (including flesh tissue, paraffin are cut
Piece, pleural effusion and whole blood).In addition, by be separately added into reaction solution 10000 copy wild template (A549) and
Then the mutated genes (G719X/S768I/L861Q) of different proportion carry out augmentation detection through the invention again to verify it
Sensitivity.
2, PCR amplification
According to the positional relationship of this project EGFR mutation type to be checked, we devise 6 pairs of primers (please correct) and are directed to
G719X/S768I/L861Q different mutation types are expanded, SEQ ID NO.10 and SEQ ID NO.11/SEQ ID
The amplified production of NO.12/SEQ ID NO.13/SEQ ID NO.14 includes G719X mutation type；SEQ ID NO.26 and SEQ
The amplified production of ID NO.27 includes S768I mutation type；The amplified production of SEQ ID NO.28 and SEQ ID NO.29 includes
L861Q mutation type.
A) synthetic pcr primer object/specific probe: synthetic method is existing conventional DNA synthetic method.
B) it prepares PCR reaction solution: preparing the PCR reaction solution of 15 microlitres/person-portion, each ingredient in everyone PCR reaction solution divided
And concentration be respectively as follows: upstream primer concentration be respectively 0.2 it is micro- rub/liter, Taq enzyme is 1U/ microlitres micro-, 1X PCR buffer, MgCl2
For 1.5 mmoles/liter, dNTP be 0.2 mmoles/liter, template DNA 1-10 nanogram/microlitre.
C) PCR amplification: the response procedures of PCR amplification are as follows: 95 DEG C initial denaturation 5 minutes；95 DEG C be denaturalized 30 seconds, 70 DEG C 20 seconds,
60 DEG C renaturation 30 seconds, 72 DEG C extend 30 seconds, recycle 35 times.Product after PCR amplification includes the DNA fragmentation of patient to be detected.
3, signal collection
Corresponding fluorescence signal is collected at 60 DEG C of the second stage in PCR amplification program.The present invention utilizes modified-
ARMS-PCR technology distinguishes wild type and mutated genes sequence, judges by the fluorescence intensity in reaction system detection knot
Fruit.VIC is internal standard signal, for detecting whether leakage plus template.
Outer mark system is used for the quality of detection template, and external control system is individually, so not influenced by double reaction, outside
The CT value of table system signal should be less than 30, and it is too low to be otherwise considered as template concentrations, and testing requirements are not achieved.
Detection architecture is for detecting whether there are mutational sites: after internal standard and external standard signal reach requirement, to detect body
Cycle-index Ct value is as judgment criteria needed for fluorescence signal in system reaches given threshold.
Fig. 3 is the testing result of EGFR gene hot spot mutation G719X/S768I/L861Q, 1. A549 copied for 10000
Under wild type gene group background, the G719X plasmid (channel ROX) of 100 copies；2. for the A549 wild type gene of 10000 copies
Under group background, the S768I plasmid (channel FAM) of 100 copies；3. under the A549 wild type gene group background for 10000 copies,
The L861Q plasmid (channel CY5) of 100 copies；4. for the A549 wild type gene VIC Air conduct measurement of 10000 copies；5. being
The A549 wild type gene group ROX Air conduct measurement of 10000 copies；6. for the channel A549 wild type gene group FAM of 10000 copies
Detection；7. for the A549 wild type gene group CY5 Air conduct measurement of 10000 copies.
Embodiment four: the detection sensitivity in various difference mutational sites
1. extracting template DNA:
Extracting template DNA from the tissue samples of patient using other commercial kits, (including flesh tissue, paraffin are cut
Piece, pleural effusion and whole blood).In addition, by be separately added into reaction solution 10000 copy wild template (A549) and
Then the mutated genes (T790M/L858R) of different proportion carry out augmentation detection through the invention again to verify its sensitivity.
2.PCR amplification
According to the positional relationship of this project EGFR mutation type to be checked, we devise 17 pairs of primers and dash forward for different
Become type to be expanded, the amplified production of SEQ ID NO.8 and SEQ ID NO.9 includes EX19DEL mutation type；SEQ ID
The amplified production of NO.10 and SEQ ID NO.11/SEQ ID NO.12/SEQ ID NO.13/SEQ ID NO.14 includes G719X
Mutation type；SEQ ID NO.15 and SEQ ID NO.16/SEQ ID NO.17/SEQ ID NO.18/SEQ ID NO.19/
The amplified production of SEQ ID NO.20/SEQ ID NO.21 includes EX20ins mutation type；SEQ ID NO.22 and SEQ ID
The amplified production of NO.23 includes L858R mutation type；The amplified production of SEQ ID NO.24 and SEQ ID NO.25 includes
T790M mutation type；The amplified production of SEQ ID NO.26 and SEQ ID NO.27 includes S768I mutation type；SEQ ID
The amplified production of NO.28 and SEQ ID NO.29 includes L861Q mutation type.
A) synthetic pcr primer object/specific probe: synthetic method is existing conventional DNA synthetic method.
B) it prepares PCR reaction solution: preparing the PCR reaction solution of 15 microlitres/person-portion, each ingredient in everyone PCR reaction solution divided
And concentration be respectively as follows: upstream primer concentration be respectively 0.2 it is micro- rub/liter, Taq enzyme is 1U/ microlitres micro-, 1X PCR buffer, MgCl2
For 1.5 mmoles/liter, dNTP be 0.2 mmoles/liter, template DNA 1-10 nanogram/microlitre.
C) PCR amplification: the response procedures of PCR amplification are as follows: 95 DEG C initial denaturation 5 minutes；95 DEG C be denaturalized 30 seconds, 70 DEG C 20 seconds,
60 DEG C renaturation 30 seconds, 72 DEG C extend 30 seconds, recycle 35 times.Product after PCR amplification includes the DNA fragmentation of patient to be detected.
3. signal collection
Corresponding fluorescence signal is collected at 60 DEG C of the second stage in PCR amplification program.The present invention utilizes modified-
ARMS-PCR technology distinguishes wild type and mutated genes sequence, judges by the fluorescence intensity in reaction system detection knot
Fruit.VIC is internal standard signal, for detecting whether leakage plus template.
Outer mark system is used for the quality of detection template, and external control system is individually, so not influenced by double reaction, outside
The CT value of table system signal should be less than 30, and it is too low to be otherwise considered as template concentrations, and testing requirements are not achieved.
Detection architecture is for detecting whether there are mutational sites: after internal standard and external standard signal reach requirement, to detect body
Cycle-index Ct value is as judgment criteria needed for fluorescence signal in system reaches given threshold.Concrete outcome is shown in Table 4
Table 4. detects the sensitivity in different mutational sites using the present invention
| Mutational site | Sensitivity | |
| 1 | L858R | 0.20% |
| 2 | T790M | 1.0% |
| 3 | S768I | 1.0% |
| 4 | G719S | Lower than 1.0% |
| 5 | G719A | 1.0% |
| 6 | G719D | Lower than 1.0% |
| 7 | G719C | 1.0% |
| 8 | L861Q | 1.0% |
| 9 | 19Del | 1.0% |
| 10 | 20insert | 1.0% |
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be within the scope of protection determined by the claims.
Sequence table
<110>Shanghai Mai Jing nanosecond science and technology Co., Ltd
<120>a kind of EGFR mutated gene detection method based on multiple fluorescence PCR
<160> 38
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tcgaggattt ccttgttggc tttcg 25
<210> 2
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
aagctctctt gaggatcttg aagga 25
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
acggtggagg tgaggcag 18
<210> 4
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tgggtgcgga agagaaagaa tacc 24
<210> 5
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ggctgcctcc tggactatgt c 21
<210> 6
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
cacgtgtgcc gcctgc 16
<210> 7
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
accgcagcat gtcaagatca cag 23
<210> 8
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
tgagaaagtt aaaattcccg tcgct 25
<210> 9
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
cacacagcaa agcagaaact cac 23
<210> 10
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
cccagcttgt ggagcctctt acacc 25
<210> 11
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
cgtgccgaac gcaccggagt 20
<210> 12
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
gtgccgaacg caccggagca 20
<210> 13
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
cgtgccgaac gcaccggagc t 21
<210> 14
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
gtgccgaacg caccggagg 19
<210> 15
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
ttctggccac catgcgaagc 20
<210> 16
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
gggttatcca cgctggccac 20
<210> 17
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
gttgtccacg ctgtccacgc 20
<210> 18
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
gcacacgtgg gggttaccgt 20
<210> 19
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
gcggcacacg tggtggggg 19
<210> 20
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
ggttgtccac gctggccacg 20
<210> 21
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
ggttgtccac gctggttacg 20
<210> 22
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
gctgacctaa agccacctcc ttac 24
<210> 23
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
tgtcaagatc acagattttg ggcg 24
<210> 24
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
ctccaccgtg carctcatca t 21
<210> 25
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
ttgagcaggt actgggagcc aat 23
<210> 26
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
ctccaggaag cctacgtgat ggccat 26
<210> 27
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
gtggaggtga ggcagatgcc 20
<210> 28
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
cttggtgcac cgcgacctg 19
<210> 29
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
tctttctctt ccgcacccag ct 22
<210> 30
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
aattaagaga agcaacat 18
<210> 31
<211> 11
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
ggagcccagc a 11
<210> 32
<211> 13
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
ttgggctggc caa 13
<210> 33
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
ctcatcacgc agctc 15
<210> 34
<211> 12
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
tggccagcgt gg 12
<210> 35
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
cacccagcag tttgg 15
<210> 36
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
ttctgacctg aaggctctgc gcg 23
<210> 37
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
agatttggac ctgcgagcg 19
<210> 38
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
gagcggctgt ctccacaagt 20
Claims (10)
1. a kind of EGFR mutated gene detection method based on multiple fluorescence PCR, which comprises the following steps:
Step 1: preparation is directed to the detection probe in the different mutational sites EGFR, it is prominent that the detection probe respectively corresponds an EGFR
Point is conjugated, detection probe described in each includes the sequence of 15-25 base, in the medium position of the sequence, the detection
The both ends of probe are marked with fluorescence detection group and quenching group respectively；
Step 2: the specific ARMS- amplimer in the corresponding different mutational sites EGFR of preparation, is reacted using multiple fluorescence PCR
System expands the segment containing mutated gene in measuring samples；
Step 3: preparation corresponds to the blocker in the different mutational sites EGFR to inhibit the amplification of wild-type genotype, recycle multiple
Fluorescence PCR system expands the segment containing mutated gene in measuring samples；
Step 4: determining measuring samples with the presence or absence of corresponding EGFR mutation type according to fluorescence signal.
2. the EGFR mutated gene detection method based on solid-phase hybridization technology as described in claim 1, which is characterized in that described
Detection probe is the DNA of at least one sequence or its reverse complementary sequence in SEQ ID NO.NO.1-7.
3. the EGFR mutated gene detection method based on multiple fluorescence PCR as described in claim 1, which is characterized in that described
Fluorescence detection group is FAM, ROX, CY5 and HEX one kind.
4. the EGFR mutated gene detection method based on multiple fluorescence PCR as described in claim 1, which is characterized in that described
Blocker in third step covers mutational site sequence, matches completely with wild type sequence, at least there is an alkali with mutant nucleotide sequence
Base mispairing.
5. a kind of EGFR mutated gene detection method based on multiple fluorescence PCR as described in claim 1, which is characterized in that
The different mutational sites EGFR are in G719X, 19-del, 20-insertions, T790M, S768I, L858R and L861Q
At least one.
6. the EGFR mutated gene detection method based on multiple fluorescence PCR as described in claim 1, which is characterized in that described
Amplimer is at least a pair of of following 17 centering: SEQ ID NO.8 and SEQ ID NO.9 in second step；SEQ ID NO.10
With SEQ ID NO.11；SEQ ID NO.10 and SEQ ID NO.12；SEQ ID NO.10 and SEQ ID NO.13；SEQ ID
NO.10 and SEQ ID NO.14；SEQ ID NO.15 and SEQ ID NO.16；SEQ ID NO.15 and SEQ ID NO.17；SEQ
ID NO.15 and SEQ ID NO.18；SEQ ID NO.15 and SEQ ID NO.19；SEQ ID NO.15 and SEQ ID NO.20；
SEQ ID NO.15 and SEQ ID NO.21；SEQ ID NO.22 and SEQ ID NO.23；SEQ ID NO.24 and SEQ ID
NO.25；SEQ ID NO.26 and SEQ ID NO.27；SEQ ID NO.28 and SEQ ID NO.29.
7. the EGFR mutated gene detection method based on multiple fluorescence PCR as claimed in claim 5, the PCR amplification primer
It can combine in different ways and different loci is carried out while being expanded in single reaction pipe: SEQ ID NO.8/SEQ ID
NO.9/SEQ ID NO.15/SEQ ID NO.16/SEQ ID NO.17/SEQ ID NO.18/SEQ ID NO.19/SEQ ID
NO.20/SEQ ID NO.21 can expand the mutation type comprising 19DEL and EX20insert simultaneously；SEQ ID NO.22/SEQ
ID NO.23/SEQ ID NO.24/SEQ ID NO.25 can expand the mutation type comprising L858R and T790M simultaneously；SEQ ID
NO.10/SEQ ID NO.11/SEQ ID NO.12/SEQ ID NO.13/SEQ ID NO.14/SEQ ID NO.26/SEQ ID
NO.27/SEQ ID NO.28/SEQ ID NO.29 can be expanded simultaneously comprising G719D/G719A/G719S/G719C/S768I/
The mutation type of L861Q；The amplified production of SEQ ID NO.37 and SEQ ID NO.38 includes internal control sequence.
8. the EGFR mutated gene detection method based on multiple fluorescence PCR as described in claim 1, which is characterized in that described
The sequence length of blocker is 10bp-25bp, is modified in the blocker series containing PNA or LNA.
9. the EGFR mutated gene detection method based on solid-phase hybridization technology as claimed in claim 8, which is characterized in that described
Blocker sequence is at least one sequence or its reverse complemental DNA sequence dna in SEQ ID NO.30-SEQID NO.35, often
One blocker corresponds to the EGFR mutated gene type described at least one, wherein SEQ ID NO.30 is corresponding prominent
For change type into 19DEL, the corresponding mutation type of SEQ ID NO.31 is G719D/G719A/G719S/G719C, SEQ ID
The corresponding mutation type of NO.32 is L858R, and the corresponding mutation type of SEQ ID NO.33 is T790M, and SEQ ID NO.34 is corresponding
Mutation type be S768I, the corresponding mutation type of SEQ ID NO.35 be L861Q.
10. a kind of kit for detecting EGFR mutated gene, it is characterised in that the kit benefit of the detection EGFR mutated gene
With being carried out including the EGFR mutated gene detection method as described in any one of claims 1-9 based on multiple fluorescence PCR
Detection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811518772.9A CN109457018A (en) | 2018-12-12 | 2018-12-12 | A kind of EGFR mutated gene detection method based on multiple fluorescence PCR |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811518772.9A CN109457018A (en) | 2018-12-12 | 2018-12-12 | A kind of EGFR mutated gene detection method based on multiple fluorescence PCR |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109457018A true CN109457018A (en) | 2019-03-12 |
Family
ID=65613093
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811518772.9A Pending CN109457018A (en) | 2018-12-12 | 2018-12-12 | A kind of EGFR mutated gene detection method based on multiple fluorescence PCR |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109457018A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110373454A (en) * | 2019-03-27 | 2019-10-25 | 杭州丹威生物科技有限公司 | A kind of kit and method of joint-detection EGFR genetic mutation |
| CN110438206A (en) * | 2019-08-23 | 2019-11-12 | 杭州迪安医学检验中心有限公司 | Primer, probe and the kit of one group of 19 Exon deletion of detection EGFR gene mutation |
| CN110923325A (en) * | 2019-12-30 | 2020-03-27 | 阅尔基因技术（苏州）有限公司 | Primer Blocker group, kit and method for detecting EGFR gene mutation |
| CN111334573A (en) * | 2020-04-28 | 2020-06-26 | 重庆浦洛通基因医学研究院有限公司 | Gene detection kit for hypertension medication and use method |
| CN111690752A (en) * | 2020-07-16 | 2020-09-22 | 重庆浦洛通基因医学研究院有限公司 | Kit and method for detecting human EGFR gene mutation |
| CN112322733A (en) * | 2020-10-28 | 2021-02-05 | 苏州中科先进技术研究院有限公司 | Nucleic acid composition and kit for detecting EGFR gene mutation and method for detecting EGFR gene mutation |
| CN114592045A (en) * | 2022-04-29 | 2022-06-07 | 江苏吉诺思美精准医学科技有限公司 | Primer and probe composition for detecting EGFR gene mutation by using multiple fluorescent pcr, kit and detection method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1144683A1 (en) * | 1999-01-19 | 2001-10-17 | Universite De Montreal | Process for the generation of oligonucleotide libraries (ols) representative of genomes or expressed mrnas (cdnas) and uses thereof |
| CN102747157A (en) * | 2012-07-16 | 2012-10-24 | 武汉海吉力生物科技有限公司 | Primers, probes, kit and method for detecting human EGFR (epidermal growth factor receptor) gene mutations |
| CN103255201A (en) * | 2012-02-16 | 2013-08-21 | 北京宏微特斯生物科技有限公司 | Method of detecting gene mutation based on Blocker primers and ARMS primers, and kit |
| CN108048531A (en) * | 2017-11-16 | 2018-05-18 | 苏州吉玛基因股份有限公司 | A kind of super retardance fluorescence quantifying PCR method of highly sensitive detection rare mutation |
| CN108949927A (en) * | 2018-08-17 | 2018-12-07 | 上海迈景纳米科技有限公司 | It is a kind of for detecting the sequence composition and its kit of human EGFR gene mutations |
-
2018
- 2018-12-12 CN CN201811518772.9A patent/CN109457018A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1144683A1 (en) * | 1999-01-19 | 2001-10-17 | Universite De Montreal | Process for the generation of oligonucleotide libraries (ols) representative of genomes or expressed mrnas (cdnas) and uses thereof |
| CN103255201A (en) * | 2012-02-16 | 2013-08-21 | 北京宏微特斯生物科技有限公司 | Method of detecting gene mutation based on Blocker primers and ARMS primers, and kit |
| CN107419018A (en) * | 2012-02-16 | 2017-12-01 | 江苏宏微特斯医药科技有限公司 | A kind of method and kit based on Blocker primers and ARMS primer detection gene mutations |
| CN102747157A (en) * | 2012-07-16 | 2012-10-24 | 武汉海吉力生物科技有限公司 | Primers, probes, kit and method for detecting human EGFR (epidermal growth factor receptor) gene mutations |
| CN108048531A (en) * | 2017-11-16 | 2018-05-18 | 苏州吉玛基因股份有限公司 | A kind of super retardance fluorescence quantifying PCR method of highly sensitive detection rare mutation |
| CN108949927A (en) * | 2018-08-17 | 2018-12-07 | 上海迈景纳米科技有限公司 | It is a kind of for detecting the sequence composition and its kit of human EGFR gene mutations |
Non-Patent Citations (2)
| Title |
|---|
| XUEFEI LI 等: "Comprehensive Analysis of EGFR-Mutant Abundance and Its Effect on Efficacy of EGFR TKIs in Advanced NSCLC with EGFR Mutations", 《J THORAC ONCOL》 * |
| 闵丽姗 等: "锁核酸扩增阻滞荧光聚合酶链反应技术检测结直肠癌患者血浆肿瘤K-ras基因突变", 《中华实验外科杂志》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110373454A (en) * | 2019-03-27 | 2019-10-25 | 杭州丹威生物科技有限公司 | A kind of kit and method of joint-detection EGFR genetic mutation |
| CN110438206A (en) * | 2019-08-23 | 2019-11-12 | 杭州迪安医学检验中心有限公司 | Primer, probe and the kit of one group of 19 Exon deletion of detection EGFR gene mutation |
| CN110923325A (en) * | 2019-12-30 | 2020-03-27 | 阅尔基因技术（苏州）有限公司 | Primer Blocker group, kit and method for detecting EGFR gene mutation |
| CN111334573A (en) * | 2020-04-28 | 2020-06-26 | 重庆浦洛通基因医学研究院有限公司 | Gene detection kit for hypertension medication and use method |
| CN111690752A (en) * | 2020-07-16 | 2020-09-22 | 重庆浦洛通基因医学研究院有限公司 | Kit and method for detecting human EGFR gene mutation |
| CN112322733A (en) * | 2020-10-28 | 2021-02-05 | 苏州中科先进技术研究院有限公司 | Nucleic acid composition and kit for detecting EGFR gene mutation and method for detecting EGFR gene mutation |
| CN114592045A (en) * | 2022-04-29 | 2022-06-07 | 江苏吉诺思美精准医学科技有限公司 | Primer and probe composition for detecting EGFR gene mutation by using multiple fluorescent pcr, kit and detection method |
| CN114592045B (en) * | 2022-04-29 | 2024-03-26 | 江苏吉诺思美精准医学科技有限公司 | Primer and probe composition for detecting EGFR gene mutation by multiple fluorescence pcr, kit and detection method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109457018A (en) | A kind of EGFR mutated gene detection method based on multiple fluorescence PCR | |
| CN104513864B (en) | For detecting the primer of human EGFR gene mutations, probe and test kit | |
| CN104818318B (en) | Primer, probe, detection architecture and the kit of disposable detection lung cancer multiple gene | |
| CN103710460B (en) | Test kit of detection by quantitative EGFR genetic mutation and uses thereof | |
| CN108949927B (en) | Sequence composition for detecting human EGFR gene mutation and kit thereof | |
| CN107447013B (en) | Method for detecting mutation sites of codons 12 and 13 of Kras gene and kit thereof | |
| CN102776286B (en) | Primer, probe and assay kit for detecting v-ros avian UR2 sarcoma viral oncogene homolog 1 (ROS1) gene fusion mutation | |
| CN110938693B (en) | Primer group, kit and method for detecting BRAF gene mutation | |
| CN111235272B (en) | Composition for once detecting multiple gene mutation of lung cancer and application thereof | |
| CN108456721B (en) | Method for synchronously detecting gene mutation and methylation and application thereof | |
| CN112824535A (en) | Primer composition for gene mutation multiplex detection and kit thereof | |
| CN111206100A (en) | Kit and method for detecting mutation of T790M site of EGFR gene | |
| CN113584132B (en) | Digital PCR (polymerase chain reaction) kit for detecting human EGFR (epidermal growth factor receptor) gene mutation | |
| CN105803076A (en) | Specific primer and probe for detecting T790M locus of EGFR gene | |
| CN110863053A (en) | Primer, probe and method for detecting EGFR vIII mutant | |
| CN109097470A (en) | A kind of EGFR mutated gene detection method based on fluorescence-encoded micro-beads | |
| CN107937524A (en) | Mankind's KRAS gene mutation detection kit and detection method | |
| CN108300785A (en) | A kind of primer combination of probe of BRAF gene mutation detection and its application | |
| CN113930500A (en) | Digital PCR (polymerase chain reaction) detection method for human PIK3CA gene mutation and application | |
| CN111304305A (en) | Kit and method for detecting EGFR gene methylation | |
| CN110066873A (en) | Specific primer, probe and kit based on the detection EGFR gene L858R mutation of digital pcr technology | |
| CN116555423A (en) | Lung cancer methylation marker combination, detection product and application thereof | |
| WO2019084998A1 (en) | Kit for detecting kras, nras, or braf gene mutation | |
| CN113930501A (en) | Digital PCR detection method for human EGFR gene mutation and application | |
| CN110684849A (en) | Primer, probe, kit and method for detecting KRAS gene mutation of human circulating tumor cell based on ddPCR |
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 | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20190312 Assignee: Joinstar Biomedical Technology Co.,Ltd. Assignor: JIANGSU NANOFE BIOMEDICAL TECH. Co.,Ltd. Contract record no.: X2020980004307 Denomination of invention: Method for detecting EGFR (epidermal growth factor receptor) mutant gene based on multiple-fluorescence PCR (polymerase chain reaction) License type: Exclusive License Record date: 20200723 |
|
| EE01 | Entry into force of recordation of patent licensing contract |