CN109182478A - A kind of digital pcr detection kit in the mutational site EGFR gene T790M - Google Patents
A kind of digital pcr detection kit in the mutational site EGFR gene T790M Download PDFInfo
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- CN109182478A CN109182478A CN201811304909.0A CN201811304909A CN109182478A CN 109182478 A CN109182478 A CN 109182478A CN 201811304909 A CN201811304909 A CN 201811304909A CN 109182478 A CN109182478 A CN 109182478A
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- fluorescence probe
- digital pcr
- egfr gene
- fluorophor
- pcr detection
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- 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
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Abstract
The invention discloses a kind of digital pcr detection kits in the mutational site EGFR gene T790M.The present invention sports test object with EGFR gene T790M, optimum organization and fluorescence probe by special primer, to realize that accurately, simply and rapidly detecting EGFR gene T790M simultaneously is mutated, and the ability of detection mutation is up to 0.1%, T790M can effectively be detected in patient's dissociative DNA to be mutated, still have for the sample short segment DNA obtained from the fixed paraffin embedding of formaldehyde and similarly expanded with fresh tissue sample DNA and detectability.
Description
Technical field
The invention belongs to molecular diagnostic techniques fields, and in particular to a kind of digital pcr in the mutational site EGFR gene T790M
Detection kit.
Background technique
In recent years, the landmark change of non-small cell lung cancer (NSCLC) therapy field is for epidermal growth factor receptor
The patients with terminal of body (EGFR) positive gene mutation is treated using EGFR- tyrosine kinase inhibitor (TKI).Treatment benefits
Patient show as no progression of disease phase (PFS) and extend, objective reactivity (ORR) is improved, and quality of life is greatly improved.To the greatest extent
Pipe is good to initial EGFR-TKI therapeutic response, but most of patient will appear after mean treatment 10 months to such drug
Drug resistance.
Early stage, 50% that there are 20 extras of EGFR gene was aobvious the study found that in the tumour being in progress after being treated using EGFR-TKI
The mutation in sub 790th site, i.e. T790M gene mutation.Therefore, clinically by detection Peripheral Blood of Patients with Non-small Cell Lung
It is mutated in dissociative DNA with the presence or absence of T790M, to judge to whether there is drug resistance after patient takes EGFR-TKI.
But often T790M mutation content is extremely low in patient's dissociative DNA, high for the susceptibility requirement of detection method,
Conventional detection method can not meet the demand well.
Summary of the invention
It is an object of the invention to overcome prior art defect, a kind of EGFR gene T790M number in mutational site is provided
PCR detection kit.
By the present invention in that using specific primer and probe in specific digital pcr reaction system, by reaction system point
Dripped at nearly 20,000 Water-In-Oils, to targeted mutagenesis amplification of nucleic acid sequences, by one with amplified production sequence can be complementary fluorescence
Probe, fluorescence probe is hydrolyzed by archaeal dna polymerase and releases fluorophor during targeted mutagenesis amplification of nucleic acid sequences, from
And the reaction oil packet water droplet number with fluorescence signal is calculated, to judge catastrophe.
Technical scheme is as follows:
A kind of digital pcr detection kit in the mutational site EGFR gene T790M, including detection primer to and fluorescence probe
Group, detection primer is to including forward primer and reverse primer as shown in SEQ ID NO 01 and 02 respectively, fluorescence probe group packet
Include first fluorescence probe as shown in SEQ ID NO 03 and 04 and the second fluorescence probe respectively, the first fluorescence probe and second glimmering
5 ' ends of light probe are marked with the first fluorophor and the second fluorophor different from each other respectively, and 3 ' ends are marked with and above-mentioned the
One and the second third fluorophor that goes out of fluorophor Xiang Hu temper, and the first fluorescence probe and the second fluorescence probe are differing from each other
Base with LNA mark,
Its reaction system is as follows:
In a preferred embodiment of the invention, first fluorophor is FAM.
In a preferred embodiment of the invention, second fluorophor is HEX.
In a preferred embodiment of the invention, the third fluorophor is BHQ.
It is further preferred that the reaction system are as follows:
The beneficial effects of the present invention are: the present invention sports test object with EGFR gene T790M, pass through special primer
Optimum organization and fluorescence probe are mutated, Er Qiejian to realize and accurately, simply and rapidly detect EGFR gene T790M simultaneously
The ability for surveying mutation is up to 0.1%, can effectively detect T790M in patient's dissociative DNA and be mutated, for from the fixed paraffin of formaldehyde
The sample of embedding short segment DNA obtained still has similarly to be expanded and detectability with fresh tissue sample DNA.
Detailed description of the invention
Fig. 1 is that digital pcr detects the negative sample detection figure of EGFR gene T790M mutation in the embodiment of the present invention 1.
Fig. 2 is that digital pcr detects the positive sample detection figure of EGFR gene T790M mutation in the embodiment of the present invention 1.
Specific embodiment
Technical solution of the present invention is further explained and described below by way of specific embodiment combination attached drawing.
Embodiment 1
Mutation standard items are prepared in the following manner: by the EGFR gene T790M mutant cell system of known mutations
Genomic DNA and wild-type cell system genomic DNA are then added in Covaris ultraphonic pipe and carry out after mixing according to different proportion
Ultrasonic treatment obtains the fragmentation DNA for interrupting and becoming 180bp close with ctDNA fragment length or so.Wild type standard items are
It obtains in the following manner: the EGFR gene T790M mutant cell system genomic DNA of known mutations is added to Covaris
It in ultraphonic pipe and is ultrasonically treated, obtains the fragmentation DNA for interrupting and becoming 180bp close with ctDNA fragment length or so.
The mutant proportion obtained according to the method described above is respectively 10%, 5% and 0.1% standard items.
The reaction system of digital pcr detection EGFR gene T790M mutation is established, the method mainly comprises the steps that
(1) the wild type DNA gene order for EGFR gene the T790M mutation and EGFR gene announced according to Cosmic data
And mutant gene sequence, design detection primer to and fluorescence probe group, detection primer is to including respectively such as 01 He of SEQ ID NO
Forward primer and reverse primer shown in 02, fluorescence probe group include first fluorescence as shown in SEQ ID NO 03 and 04 respectively
Probe and the second fluorescence probe, 5 ' ends of the first fluorescence probe are marked with FAM, and 5 ' ends of the second fluorescence probe are marked with HEX, the
3 ' the ends label of one fluorescence probe and the second fluorescence probe has, and the first fluorescence probe and the second fluorescence probe are each other not
Identical base is marked with LNA, specially following first fluorescence probe T790M-M-P-FAM and the second fluorescence probe T790M-
The base of capitalization overstriking font in WT-P-HEX is marked with LNA:
T790M-M-P-FAM:tgagctgcGtgatgag
T790M-WT-P-HEX:tgagctgcAtgatgag.
(2) preparation of digital pcr amplification reaction system and upper machine testing.
(3) fluorescence signal is detected, with the standard for thering is the droplet hole count of fluorescence signal to judge as a result.
The EGFR gene T790M wildtype gene sequence and mutation mutation base announced in step (1) according to Cosmic data
Because of sequence, high specific Mdification primer and specificity fluorescent probe are designed, following table is detailed in.
The digital pcr amplification reaction system of step (2) are as follows:
| Serial number | Material | Material concentration | Dosage (μ L) |
| 1 | 10 × PCR buffer | 10× | 5 |
| 2 | MgCl2 | 25mM | 4 |
| 3 | dNTPs | 10mM | 6 |
| 4 | T790M-F(SEQ ID NO 01) | 50μM | 2 |
| 5 | T790M-R(SEQ ID NO 02) | 50mM | 2 |
| 6 | T790M-M-P-FAM(SEQ ID NO 03) | 50mM | 2 |
| 7 | T790M-WT-P-HEX(SEQ ID NO 04) | 50mM | 2 |
| 8 | Taq enzyme | 5U | 1 |
| 9 | H2O | Purified water | 21 |
| 10 | DNA profiling | 2ng/ul | 5 |
| 11 | Total volume | 50 |
The above reagent component: 10 × PCR buffer, MgCl2, Taq enzyme, dNTP is purchased from Sai Mofei company.
The reaction condition of the fluorescent PCR is 95 DEG C of initial denaturations 10 minutes, 1 circulation；94 DEG C are denaturalized 30 seconds, and 60 DEG C are moved back
Fire/extension 1 minute, 40 circulations；98 DEG C are moved back enzyme activity 10 minutes, 4 DEG C of preservations.
Step (3) detects FAM and HEX fluorescence signal, is using Bio-rad QX-200 digital pcr amplification instrument (reactant
System's processing reference
https://wenku.baidu.com/view/81cbe361e418964bcf84b9d528ea81c758f52e
07.html).Primary detectable 96 parts of samples (including yin and yang attribute control).Sentenced according to the positive hole count that fluorescent PCR amplification instrument is shown
Disconnected result: FAM the and HEX fluorescence intensity of reaction system is detected, shows that loading amount of DNA exists when reaching given threshold with HEX signal
In allowed band, FAM signal results are credible；Reach judging as yin and yang attribute for the threshold line of setting or more using FAM signal hole count
Standard, mutant proportion is FAM signal hole count/(FAM signal hole count+HEX signal hole count).
Fig. 1 is that digital pcr of the present invention detects the negative sample detection figure of EGFR gene T790M mutation；Fig. 2 is number of the present invention
Word PCR detects the positive sample detection figure of EGFR gene T790M mutation；
Embodiment 2
A kind of digital pcr detection kit in the mutational site EGFR gene T790M, including detection primer to and fluorescence probe
Group, detection primer is to including forward primer and reverse primer as shown in SEQ ID NO 01 and 02 respectively, fluorescence probe group packet
Include first fluorescence probe as shown in SEQ ID NO 03 and 04 and the second fluorescence probe respectively, the first fluorescence probe and second glimmering
5 ' ends of light probe are marked with the first fluorophor and the second fluorophor different from each other respectively, and 3 ' ends are marked with and above-mentioned the
One and the second third fluorophor that goes out of fluorophor Xiang Hu temper, and the first fluorescence probe and the second fluorescence probe are differing from each other
Base with LNA mark,
Its reaction system is as follows:
First fluorophor is FAM.Second fluorophor is HEX.The third fluorophor is BHQ.
The foregoing is only a preferred embodiment of the present invention, the range that the present invention that therefore, it cannot be limited according to is implemented, i.e.,
Equivalent changes and modifications made in accordance with the scope of the invention and the contents of the specification should still be within the scope of the present invention.
Sequence table
<110>Xiamen Fei Shuo Bioisystech Co., Ltd
<120>the digital pcr detection kit in the mutational site a kind of EGFR gene T790M
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ctctccctcc ctccagga 18
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ttgtgttccc ggacatagtc 20
<210> 3
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tgagctgcgt gatgag 16
<210> 4
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tgagctgcat gatgag 16
Claims (5)
1. a kind of digital pcr detection kit in the mutational site EGFR gene T790M, it is characterised in that: including detection primer pair
With fluorescence probe group, detection primer is glimmering to including forward primer and reverse primer as shown in SEQ ID NO 01 and 02 respectively
Light probe group includes that first fluorescence probe as shown in SEQ ID NO 03 and 04 and the second fluorescence probe, the first fluorescence are visited respectively
5 ' ends of needle and the second fluorescence probe are marked with the first fluorophor and the second fluorophor different from each other, 3 ' end labels respectively
There are the third fluorophor to go out with above-mentioned first and second fluorophors Xiang Hu temper, and the first fluorescence probe and the second fluorescence probe
Base differing from each other is marked with LNA,
Its reaction system is as follows:
2. digital pcr detection kit as described in claim 1, it is characterised in that: first fluorophor is FAM.
3. digital pcr detection kit as described in claim 1, it is characterised in that: second fluorophor is HEX.
4. digital pcr detection kit as described in claim 1, it is characterised in that: the third fluorophor is BHQ.
5. the digital pcr detection kit as described in any claim in Claims 1-4, it is characterised in that: the reaction
System are as follows:
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811304909.0A CN109182478A (en) | 2018-11-02 | 2018-11-02 | A kind of digital pcr detection kit in the mutational site EGFR gene T790M |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811304909.0A CN109182478A (en) | 2018-11-02 | 2018-11-02 | A kind of digital pcr detection kit in the mutational site EGFR gene T790M |
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| CN109182478A true CN109182478A (en) | 2019-01-11 |
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| CN201811304909.0A Pending CN109182478A (en) | 2018-11-02 | 2018-11-02 | A kind of digital pcr detection kit in the mutational site EGFR gene T790M |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112210606A (en) * | 2020-11-17 | 2021-01-12 | 远辰生物科技(苏州)有限公司 | Digital PCR detection system for EGFR T790M locus and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591472A (en) * | 2017-01-11 | 2017-04-26 | 北京泛生子基因科技有限公司 | Kit, reaction system and method for detecting human EGFR gene T790M mutation |
| CN107083438A (en) * | 2017-06-12 | 2017-08-22 | 上海捷易生物科技有限公司 | Detect primer, probe and the method for EGFR gene 20 extron T790M and C797S mutation |
| CN107488728A (en) * | 2017-09-26 | 2017-12-19 | 臻和（北京）科技有限公司 | Primer combination of probe thing, kit and the method for 3D digital pcrs detection EGFR specific gene mutation |
| CN108517359A (en) * | 2017-02-24 | 2018-09-11 | 北京创新乐土生物科技有限公司 | The kit of EGFR gene T790M mutation is detected in patients with lung cancer |
-
2018
- 2018-11-02 CN CN201811304909.0A patent/CN109182478A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591472A (en) * | 2017-01-11 | 2017-04-26 | 北京泛生子基因科技有限公司 | Kit, reaction system and method for detecting human EGFR gene T790M mutation |
| CN108517359A (en) * | 2017-02-24 | 2018-09-11 | 北京创新乐土生物科技有限公司 | The kit of EGFR gene T790M mutation is detected in patients with lung cancer |
| CN107083438A (en) * | 2017-06-12 | 2017-08-22 | 上海捷易生物科技有限公司 | Detect primer, probe and the method for EGFR gene 20 extron T790M and C797S mutation |
| CN107488728A (en) * | 2017-09-26 | 2017-12-19 | 臻和（北京）科技有限公司 | Primer combination of probe thing, kit and the method for 3D digital pcrs detection EGFR specific gene mutation |
Non-Patent Citations (2)
| Title |
|---|
| KENNETH S THRESS等: "Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M", 《NAT MED.》 * |
| 黄斐等: "数字PCR检测非小细胞肺癌患者血浆EGFR基因T790M突变平台的建立", 《中华检验医学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112210606A (en) * | 2020-11-17 | 2021-01-12 | 远辰生物科技(苏州)有限公司 | Digital PCR detection system for EGFR T790M locus and application thereof |
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Application publication date: 20190111 |