CN108517359A - The kit of EGFR gene T790M mutation is detected in patients with lung cancer - Google Patents
The kit of EGFR gene T790M mutation is detected in patients with lung cancer Download PDFInfo
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- CN108517359A CN108517359A CN201710101976.1A CN201710101976A CN108517359A CN 108517359 A CN108517359 A CN 108517359A CN 201710101976 A CN201710101976 A CN 201710101976A CN 108517359 A CN108517359 A CN 108517359A
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- 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
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Abstract
The invention discloses a kind of kits for detecting EGFR gene T790M mutation in patients with lung cancer.Kit provided by the present invention for detecting EGFR gene T790M mutation in patients with lung cancer contains two ssDNA probes；Two ssDNA probes are ssDNA probe first and ssDNA probe second；The sequence of the ssDNA probe first is sequence 3 in sequence table；The sequence of the ssDNA probe second is sequence 4 in sequence table.EGFR gene T790M mutation are detected in patients with lung cancer using kit of the present invention, accuracy rate of testing result is very high, and the testing result provided can be used for auxiliary diagnosis or instruct clinical application scheme etc..
Description
Technical field
The invention belongs to biomedicine fields, are related to a kind of for detecting EGFR gene T790M mutation in patients with lung cancer
Kit.
Background technology
Cancer is one of most important non-communicable diseases in the whole world, and the highest chronic disease of lethality at present, according to me
State《Cancer in China statistics in 2015》, China increases 429.2 ten thousand cases of cancer newly in 2015, cancer mortality case or is more than
281.4 ten thousand.Wherein lung cancer is the highest cancer kind of morbidity and mortality.
Surface growth factor receptors (epidermal growth factor receptor, EGFR) are important lung cancer drive
Dynamic gene, EGFR-TKIs are the micromolecular inhibitor for this target spot, clinical research confirmation, the evening of EGFR gene hot spot mutation
Phase Patients with Non-small-cell Lung can significantly benefit from EGFR-TKI, but be more than that 80% patient took EGFR-TKI in 1 year
Drug resistance may be developed, about 50% drug resistant patient has the 790th bit codon missense mutation of 20 exons of EGFR (i.e.
T790M is mutated).And AstraZeneca has developed new targeting medicine specifically for the site -- difficult to understand this replaces Buddhist nun, it is one novel
Tyrosine kinase inhibitor class new drug, can efficiently, specifically inhibit the advanced lung cancer for having occurred and that drug resistance, and obtain
The certification of FDA.
It is current to have two kinds of methods, real-time fluorescence quantitative PCR (Real Time PCR) base by the gene quantification of round pcr
It is a kind of method of relative quantification in Ct values；Digital pcr (Digital PCR, dPCR) is carried out based on single-molecule PCR method
It counts, is a kind of method of absolute quantitation.DPCR as third generation round pcr, any caliberator or external standard can not depended on and
It is using directly counting target molecule, you can the absolute number for determining target molecule is the absolute quantitation to initial sample.Therefore
Especially suitable for the application field that cannot be differentiated very well by Ct values, such as：Copy number variation, abrupt climatic change, gene relative expression
Research (such as allele imbalance is expressed), the verification of two generation sequencing results, miRNA expression analysis, unicellular gene expression analysis
Deng.Although present genetic test industry clinically uses also more with caution dPCR, there are many researchers
The quantitative detection of DNA is used it for, and multinomial result of study also turns out that dPCR technologies apply the feasibility in the field.These
Research is related to the prognosis evaluation of tumour, all directions such as outcome prediction and real time monitoring.
Invention content
The object of the present invention is to provide a kind of kits for detecting EGFR gene T790M mutation in patients with lung cancer.
Kit provided by the present invention for detecting EGFR gene T790M mutation in patients with lung cancer contains two
SsDNA probe；Two ssDNA probes are that ssDNA probe first (mutant probe) and ssDNA probe second are (wild
Probe)；The sequence of the ssDNA probe first is sequence 3 in sequence table；The sequence of the ssDNA probe second is sequence table
Middle sequence 4.
Wherein, 5 ' the ends label of the ssDNA probe first and the ssDNA probe second has,
3 ' ends label has.
In the present invention, the fluorescent reporter group of 5 ' end labels of the ssDNA probe first (mutant probe) is specially
The quenching group of FAM, 3 ' end labels are specially MGB；The fluorescent reporter group of the end of DNA probe second (wild probe) 5 ' label
The quenching group of specially VIC, 3 ' end labels are specially MGB.
As needed, primer pair there are one can also containing in the kit；Sense primer in the primer pair can basis
The upstream sequence in the mutational sites EGFR gene T790M is designed；Downstream primer in the primer pair can be according to EGFR gene
The downstream sequence in the mutational sites T790M is designed.
In the present invention, the sequence of the sense primer in the primer pair is specially sequence 1 in sequence table；The primer pair
In the sequence of downstream primer be specially sequence 2 in sequence table.
The kit is the kit based on digital pcr technology.
In the present invention, when carrying out digital pcr using the kit, the annealing temperature used is 56.0 DEG C, 58.0 DEG C
Or 60.0 DEG C.Specific response procedures are referring to embodiment.
The kit it is following it is any in application also belong to protection scope of the present invention：
(A) product that T790M mutation whether occur for detecting patients with lung cancer EGFR gene prepared；
(B) it prepares and takes whether EGFR-TKI can occur drug resistant product for detecting patients with lung cancer.
The present invention also protects the following two kinds product containing the kit：For whether detecting patients with lung cancer EGFR gene
The product that T790M mutation occur, takes whether EGFR-TKI can occur drug resistant product for detecting patients with lung cancer.
In the present invention, the lung cancer is specially non-small cell lung cancer.
It is demonstrated experimentally that using provided by the present invention prominent for detecting lung cancer EGFR gene T790M based on digital pcr technology
The kit and detection method of change, accuracy rate of testing result is very high, and the testing result provided for example can be used for auxiliary diagnosis
Or instruct clinical application scheme.
Description of the drawings
Fig. 1 is commercialization T790M probes and the experimental result of the invention from synthesising probing needle under 56.0 DEG C of annealing temperatures.Its
In, A is the experimental result that T790M probes are commercialized；B is experimental result of the present invention from synthesising probing needle.
Fig. 2 is commercialization T790M probes and the experimental result of the invention from synthesising probing needle under 58.0 DEG C of annealing temperatures.Its
In, A is the experimental result that T790M probes are commercialized；B is experimental result of the present invention from synthesising probing needle.
Fig. 3 is commercialization T790M probes and the experimental result of the invention from synthesising probing needle under 60.0 DEG C of annealing temperatures.Its
In, A is the experimental result that T790M probes are commercialized；B is experimental result of the present invention from synthesising probing needle.
In Fig. 1-Fig. 3, " red " represents wild type fluorescence, and " indigo plant " represents saltant type fluorescence, and " green " represents wild type and mutation
Type fluorescence exists simultaneously, and " Huang " represents no fluorescence.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
EGFR T790M standard items：HorizonDx Products, catalog number HD850.Wherein, gene mutation
Frequency is 1%；Product type is DNA.
Embodiment 1, the design for being used to detect primer, probe that lung cancer EGFR gene T790M is mutated based on digital pcr technology
With synthesis
According to the 790th bit codon missense mutation of 20 exons of EGFR (i.e. T790M mutation), this hair is designed and synthesized
The bright primer and probe for being used to detect the T790M mutation of lung cancer EGFR gene based on digital pcr technology, particular sequence are as follows：
Primer sequence：
Forward primer：5 '-GGCATCTGCCTCACCTCC-3 ' (sequence 1)；
Reverse primer：5 '-GACATAGTCCAGGAGGCAGC-3 ' (sequence 2).
Probe sequence：
Mutant probe：5 '-FAM-GAGCTGCATGATGAG-MGB-3 ' (sequence 3)；
Wild probe：5 '-VIC-GAGCTGCGTGATGAG-MGB-3 ' (sequence 4).
Embodiment 2 is mutated based on digital pcr technology detection lung cancer EGFR gene T790M
Test sample：EGFR T790M standard items (gene mutation frequency 1%).
One, experimental method
1, reaction solution is prepared
PCR reaction mixtures are prepared in 0.2ml PCR pipes：
Wherein, two primers and each 0.5 μ L of two probes, the final concentration of two primers and two probes in the reaction system
It is 0.3 μM.QuantStudioTM3D Digital PCR Master Mix v2 are TheromFisher Products,
Catalog number is A26358.
3, loading
The QuantStudio 3D Digital PCR Chip that the instrument used produces for ThermoFisher companies
Loader, model：237281048.
(1) chip, brush head and chip lid are placed in by step on chip loader, it is mixed from above-mentioned prepared reaction
It closes in liquid and draws 14.5 μ L, slowly squeeze into the well of brush head lower part.
(2) and then loading buttons (black strip) are pressed, chip loader can be uniform by the reaction solution in brush head
Ground is applied on chip, and during smearing, instrument green indicator light flickers, and after smearing, indicator light becomes to be always on.
(3) it after brush head smearing finishes and leaves chip, takes oil sealing needle that oil sealing about 10~15 is vacantly added dropwise at once and drops to core
In piece, it is desirable that oil sealing just covers chip surface.
(4) chip loader arms are turned so that on chip lid lid to chip, pressed at full tilt with hand 30 seconds with
On, the Lid Nest buttons of arm front are then pinned, chip loader arms are lifted and put back to original position.
(5) chip is taken out, by Chip Vertical, opens the top half of chip lid, can see an aperture, keeps aperture
In the top, syringe, at 45 degree of angles, slowly pushes syringe with chip, and oil sealing is made to be full of entire chip interior,
(6) chip is kept flat on the table, the paster that chip covers is taken off, chip lid is pressed, lid and chip are made
It clings.
4, PCR reacts
The ProFlex Base that the instrument used produces for ThermoFisher companies, model：297805586.
The chip made is positioned symmetrically in PCR instrument, heat insulating mattress on pad closes the lid, set by following procedure
Response procedures, the PCR that brings into operation reactions；
Wherein, X DEG C is 56.0 DEG C, 58.0 DEG C or 60.0 DEG C.
5, initial data analysis
The QuantStudio 3D Digital PCR System that the instrument used produces for ThermoFisher companies,
Model：237271052.
Before data analysis, first QS 3D fluorescence detectors are opened and are preheated, and are inserted into the USB flash disk of storage, instrument can be certainly
Dynamic detection USB flash disk and input data.After the completion of waiting for PCR reactions, chip is taken out from PCR instrument, then with absolute ethyl alcohol by chip list
The oil stain wiped clean in face, after chip is put into, instrument can read data and chip number automatically.The analysis feelings of every chip data
From condition can be carried out from run history are clicked in main interface, illustrate that data are not divided also when analysis state is processing
It has been analysed that, illustrate that data analysis is completed and exported into flash disk when being shown as succeed, if being shown as export
Failure then illustrates that analysis is completed but exports to USB flash disk and fails, and can click export buttons and export data.When all cores
After the data analysis of piece is completed and successfully exported, USB flash disk can be extracted, and carry out subsequent analysis.
6、QuantStudioTM 3D AnalysisSuiteTMSoftware secondary datas are analyzed
Yellow dots region is the aperture (i.e. emptying aperture, no detection sample) of no FAM or VIC fluorescence signals, red point portion in figure
It is divided into the aperture (wild sample) for sending out VIC (wild probe) fluorescence, blue portion is send out FAM (mutant probe) fluorescence small
Hole (mutagenic samples), green point are the aperture (mutation and wild sample) for existing simultaneously VIC and FAM fluorescence.
The gene mutation frequency of sample to be tested is detected with the ratio of total copy number by the copy number of mutagenic samples.
7, comparative example
Using TheromFisher companies commercial prod (catalog number 4331349-Cosmic6240) as this hair
Bright comparative example.Concrete operations are：With in the commercial kit T790M probes and primer substitute the spy in the present invention respectively
Needle (sequence 3 and sequence 4) and primer (sequence 1 and sequence 2), remaining all operation is the same as above step 1-6.
Two, experimental result
Under 56.0 DEG C of annealing temperatures, commercialization T790M probes and experimental result such as Fig. 1 institutes of the invention from synthesising probing needle
Show；Under 58.0 DEG C of annealing temperatures, the experimental result of commercialization T790M probes and the present invention from synthesising probing needle is as shown in Figure 2；60.0
Under DEG C annealing temperature, the experimental result of commercialization T790M probes and the present invention from synthesising probing needle is as shown in Figure 3.
The commercialization EGFR T790M standard items for the use of gene mutation frequency being 1% are detected, and are as a result shown：The present invention
The testing result of the gene mutation frequency of the probe of designed, designed is 1% or so, but T790M probes are commercialized by wild fluorescence
Interpretation is existed simultaneously at wild and mutation fluorescence, and it is 1% to need the value for adjusting collection of illustrative plates that can just obtain gene mutation frequency
As a result, this can participate in artifact, the inaccuracy of experiment is caused.In addition, primer can improve expansion by adjusting annealing temperature
Synergy fruit, present invention employs different annealing temperatures can obtain accurate result, therefore can more embody the present invention from design
Probe effect be better than commercialization T790M probes.
<110>Innovate land of happiness Gene Tech. Company Limited in Beijing
<120>The kit of EGFR gene T790M mutation is detected in patients with lung cancer
<130> GNCLN170431
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 1
ggcatctgcc tcacctcc 18
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 2
gacatagtcc aggaggcagc 20
<210> 3
<211> 15
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 3
gagctgcatg atgag 15
<210> 4
<211> 15
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 4
gagctgcgtg atgag 15
Claims (10)
1. the kit for detecting EGFR gene T790M mutation in patients with lung cancer, contains two ssDNA probes；It is described
Two ssDNA probes are ssDNA probe first and ssDNA probe second；The sequence of the ssDNA probe first is sequence table
Middle sequence 3；The sequence of the ssDNA probe second is sequence 4 in sequence table.
2. kit according to claim 1, it is characterised in that：The ssDNA probe first and the ssDNA probe
5 ' the ends label of second has, and 3 ' ends label has.
3. kit according to claim 2, it is characterised in that：The fluorescence of 5 ' end labels of the ssDNA probe first
Reporter group is FAM, and the quenching group of 3 ' end labels is MGB；The fluorescent reporter group of the DNA probe second 5 ' end label is
The quenching group of VIC, 3 ' end labels are MGB.
4. according to any kit in claim 1-3, it is characterised in that：Primer there are one also containing in the kit
It is right；Sense primer in the primer pair is designed according to the upstream sequence in the mutational sites EGFR gene T790M；The primer
The downstream primer of centering is designed according to the downstream sequence in the mutational sites EGFR gene T790M.
5. kit according to claim 4, it is characterised in that：The sequence of sense primer in the primer pair is sequence
Sequence 1 in table；The sequence of downstream primer in the primer pair is sequence 2 in sequence table.
6. kit according to any one of claims 1-4, it is characterised in that：The kit is based on digital pcr skill
The kit of art.
7. in claim 1-6 any kit it is following it is any in application：
(A) product that T790M mutation whether occur for detecting patients with lung cancer EGFR gene prepared；
(B) it prepares and takes whether EGFR-TKI can occur drug resistant product for detecting patients with lung cancer.
8. a kind of product that T790M mutation whether occurring for detecting patients with lung cancer EGFR gene, contains in claim 1-6
One kit.
9. one kind taking whether EGFR-TKI can occur drug resistant product for detecting patients with lung cancer, containing in claim 1-6
Any kit.
10. according to any kit or application or product in claim 1-9, it is characterised in that：The lung cancer is non-
Small Cell Lung Cancer.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710101976.1A CN108517359A (en) | 2017-02-24 | 2017-02-24 | The kit of EGFR gene T790M mutation is detected in patients with lung cancer |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710101976.1A CN108517359A (en) | 2017-02-24 | 2017-02-24 | The kit of EGFR gene T790M mutation is detected in patients with lung cancer |
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| CN108517359A true CN108517359A (en) | 2018-09-11 |
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| CN201710101976.1A Pending CN108517359A (en) | 2017-02-24 | 2017-02-24 | The kit of EGFR gene T790M mutation is detected in patients with lung cancer |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182478A (en) * | 2018-11-02 | 2019-01-11 | 厦门飞朔生物技术有限公司 | A kind of digital pcr detection kit in the mutational site EGFR gene T790M |
| CN111206100A (en) * | 2020-02-28 | 2020-05-29 | 宁波胤瑞生物医学仪器有限责任公司 | Kit and method for detecting mutation of T790M site of EGFR gene |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008228590A (en) * | 2007-03-16 | 2008-10-02 | Okayama Univ | Human non-small cell lung cancer cell line having acquired resistance to gefitinib and its use |
| CN105838777A (en) * | 2015-01-13 | 2016-08-10 | 上海张江转化医学研发中心有限公司 | Method for monitoring secondary drug resistance of lung cancer patient to tyrosine kinase inhibitor through ddPCR technology |
-
2017
- 2017-02-24 CN CN201710101976.1A patent/CN108517359A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008228590A (en) * | 2007-03-16 | 2008-10-02 | Okayama Univ | Human non-small cell lung cancer cell line having acquired resistance to gefitinib and its use |
| CN105838777A (en) * | 2015-01-13 | 2016-08-10 | 上海张江转化医学研发中心有限公司 | Method for monitoring secondary drug resistance of lung cancer patient to tyrosine kinase inhibitor through ddPCR technology |
Non-Patent Citations (3)
| Title |
|---|
| WENXIAN WANG等: "A Comparison of ddPCR and ARMS for detecting EGFR T790M status in ctDNA from advanced NSCLC patients with acquired EGFR-TKI resistance", 《CANCER MEDICINE》 * |
| 李静等: "非小细胞肺癌EGFR-TKI耐药机制及耐药后治疗策略", 《医学研究生学报》 * |
| 林万明等: "《PCR技术操作和应用指南》", 30 September 1993, 人民军医出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182478A (en) * | 2018-11-02 | 2019-01-11 | 厦门飞朔生物技术有限公司 | A kind of digital pcr detection kit in the mutational site EGFR gene T790M |
| CN111206100A (en) * | 2020-02-28 | 2020-05-29 | 宁波胤瑞生物医学仪器有限责任公司 | Kit and method for detecting mutation of T790M site of EGFR gene |
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