CN109852676A - A kind of method and kit of detection human EGFR gene T790M and L858R mutation - Google Patents
A kind of method and kit of detection human EGFR gene T790M and L858R mutation Download PDFInfo
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Abstract
The present invention provides a kind of primer and probe combination of digital pcr detection architecture for human EGFR gene T790M and L858R double mutations, a kind of digital pcr detection architecture for human EGFR gene T790M and L858R double mutations, a kind of a kind of detection method of the digital pcr of the digital pcr detection kit and human EGFR gene T790M and L858R double mutations of human EGFR gene T790M and L858R double mutations.Pass through the method for digital PCR platform in dual system, realize that the high sensitivity simultaneously for T790M and L858R detects, reduce cost, reduce the consumption of sample, particularly with the more rare ctDNA of sample, also facilitate the detection of other sample types such as tumor tissues, provide certain medication guide for the patient of non-small cell lung cancer.
Description
Technical field
The invention belongs to bio-medical technology fields, are used for human EGFR gene T790M and L858R more particularly to one kind
The primer and probe of the digital pcr detection architecture of double mutations combines, and one kind is bis- for human EGFR gene T790M and L858R
The digital pcr detection architecture being mutated again, a kind of digital pcr detection reagent of human EGFR gene T790M and L858R double mutations
The detection method of the digital pcr of box and a kind of human EGFR gene T790M and L858R double mutations.
Background technique
For a long time, lung cancer is one of highest malignant tumour of morbidity and mortality in world wide.World health group
Knit the cancer report display of International Agency for Research on Cancer publication: the new cases of global lung cancer already exceed 1,600,000, and the death rate can
Reach 1,380,000, account for the 13% and 18% of Cancer Mortality and the death rate, occupy the first in malignant tumour.Rather than
80% or more of Small Cell Lung Cancer (non-small cell lung cancer, NSCLC) Zhan Suoyou Lung Cancer Types, more than 70%
Patient middle and advanced stage has been in when making a definite diagnosis.Although the diagnostic and therapeutic method of lung cancer is being constantly progressive, the death of lung cancer
Rate is not effectively controlled still, and 5 years survival rates of patient are also less than 20%.
Human epidermal growth factor acceptor family (epidermal growth factor receptor family,
EGFR) by highly conserved tyrosine kinase area intracellular, extracellular ligand binding domain and single transmembrane district's groups at working as ligand binding domain
After ligand binding, EGF-R ELISA can be converted into dimer by monomer, so that itself phosphorus of intracorporal tyrosine kinase
Acidification further activates the signal path in downstream, to eventually lead to the proliferation and existence of cell.
The EGFR such as Gefitinib, Tarceva tyrosine kinase inhibitor (EGFR-TKI) is mutated advanced NSCLC in EGFR
The curative effect to attract people's attention is achieved in the treatment of patient.EGFR-TKI is the molecular targeted agents for EGFR, mainly by with
ATP competitive binding is located at the EGFR tyrosine kinase catalytic domain binding site of cell surface, disabling signal to it is intracellular into
The transmitting of one step inhibits growth of tumour cell and induces its apoptosis.The EGFR-TKI such as Tarceva, Gefitinib are used extensively at present
In clinic.Molecular biology research has proven to the lung carcinoma cell that there is EGFR to be mutated and treats sensitivity to EGFR-TKI.
L858R is the important mutant form of one kind of EGFR, and mutation occurs mainly in 858 smart ammonia of 21 exons
Acid mutation is leucine, this mutational site enhances close to die body highly conserved in kinase activation ring (activation loop)
Sensibility of the tumour cell to EGFR-TKI, such mutation account for 40%.T790M is the main mutant form of another kind of EGFR
Formula is that secondary mutation has occurred in application EGFR-TKI therapeutic process in the 20th exon of EGFR gene, leads to EGFR 790
Threonine on point is replaced methionine.Once threonine is replaced by methionine, result introduces 1 on the site
The bigger amino acid side chain of item constitutes steric hindrance, and the formation of the steric hindrance influences hydrogen between tyrosine kinase and EGFR-TKI
The formation of key, eventually leading to EGFR-TKI can not combine with tyrosine kinase.EGFR- is not being applied in some researcher's discoveries
In the NSCLC patient of TKI treatment, T790M mutation rate < 0.1%, and occur in the secondary drug resistance person of EGFR-TKI about 50%
T790M mutation, the research achievement reflect the secondary mutation of EGFR gene in the secondary drug resistance of EGFR-TKI from another side
Status and effect, i.e. the mutation of T790M is the most important factor of EGFR-TKI secondary resistance.
Digital pcr (Digital PCR-dPCR) technology is a kind of new detection of nucleic acids and quantitative approach, with traditional quantitative
PCR (qPCR) technology is different, and digital pcr is by the way of absolute quantitation, independent of standard curve and sample for reference, directly examines
Survey the copy number of target sequence.Since this detection mode has sensitivity, specificity and the essence outstanding than traditional qPCR
True property, dPCR are widely used rapidly, this technology rare mutation under the detection of denier sample of nucleic acid, complex background is examined
Survey and the advantage that shows of expression quantity fine difference identification aspect be commonly recognized, and its gene expression research,
MicroRNA research, genome copy numbers identification, cancer markers rare mutation detection, pathogenic microorganisms identification, transgenosis at
It broad prospect of application that all various aspects such as point identification, NGS sequencing library accurate quantification and result verification have and has been subjected to
More and more concerns.QuantStudio especially used in the present inventionTM3D digital pcr technology, is received using highdensity
Up-flow control chip technology, sample are evenly distributed into 20,000 individual reacting holes.In entire workflow, between sample
It keeps completely isolated, sample cross contamination can be effectively prevented, reduce pipetting processes, simplify operating procedure.Chip type simultaneously
Design avoids the pipeline blockage problem that droplet type system may face.
Summary of the invention
It is an object of the present invention to provide a kind of numbers for human EGFR gene T790M and L858R double mutations
The primer and probe of PCR detection architecture combines.
It is a further object to provide a kind of numbers for human EGFR gene T790M and L858R double mutations
Word PCR detection architecture.
It is a further object to provide a kind of numbers for human EGFR gene T790M and L858R double mutations
Word PCR detection kit.
In the present invention, the digital pcr can be QuantStudioTM3D digital pcr.
According on one side, the present invention provides a kind of numbers for human EGFR gene T790M and L858R double mutations
The primer and probe of word PCR detection architecture combines, and the primer and probe combination includes: the primer and probe group for T790M
It closes and the primer and probe for L858R combines,
Wherein, the primer and probe for T790M combines are as follows:
T790M upstream primer: 5 '-GCCTGCTGGGCATCTG-3 ' (SEQ ID NO.1)
T790M downstream primer: 5 '-TTTGTGTTCCCGGACATAGTC-3 ' (SEQ ID NO.2)
T790M saltant type probe: 5 '-FAM-TGAGCTGCATGATGA-MGB-NFQ-3 ' (SEQ ID NO.3)
T790M wild-type probe: 5 '-VIC-TGAGCTGCGTGATGA-MGB-NFQ-3 ' (SEQ ID NO.4),
Wherein, the primer and probe for L858R combines are as follows:
L858R upstream primer: 5 '-CGCAGCATGTCAAGATCACAGAT-3 ' (SEQ ID NO.5)
L858R downstream primer: 5 '-CTCCTTCTGCATGGTATTCTTTCTC-3 ' (SEQ ID NO.6)
L858R saltant type probe: 5 '-FAM-AGTTTGGCCCGCCCAA-MGB-NFQ-3 ' (SEQ ID NO.7)
L858R wild-type probe: 5 '-VIC-AGTTTGGCCAGCCCAA-MGB-NFQ-3 ' (SEQ ID NO.8).
Wherein FAM and VIC is fluorescence marker groups common in this field.MGB-NFQ is common in this field is quenched
Group.
According on the other hand, the present invention provides a kind of for human EGFR gene T790M and L858R double mutations
Digital pcr detection architecture, it includes the combinations of above-mentioned primer and probe.
Preferably, in digital pcr detection architecture of the invention, each probe primer concentration ratio of T790M and L858R are set
Signal value distribution both to make distinguishes, and the different probe of different probe and primer concentration, L858R to T790M and draws
The relationship of the combination of different probe primer concentration ratio and the distribution situation of signal value between object concentration and both of the above carries out
Adjustment.It is highly preferred that the digital pcr detection architecture for human EGFR gene T790M and L858R double mutations of the invention
In, the working concentration of the upstream and downstream primer of T790M and L858R is all 900nM, the wild type of T790M and the work of saltant type probe
Making concentration is 90nM, and the wild type of L858R and the working concentration of saltant type probe are 450nM.
Preferably, digital pcr detection architecture of the invention further comprises: digital pcr reaction premixed liquid；DNA profiling；
ddH2O。
Particularly, the present invention provides the digital pcr detections for human EGFR gene T790M and L858R double mutations
System comprising:
According on the other hand, the present invention provides a kind of for human EGFR gene T790M and L858R double mutations
Digital pcr detection kit, it includes the combinations of above-mentioned primer and probe.
In an embodiment of above-mentioned kit, the kit further includes the premixed liquid of digital pcr reaction, example
Such as, QuantStudioTM 3D Digital PCR Master Mix v2。
In an embodiment of above-mentioned kit, the working concentration of the upstream and downstream primer of T790M and L858R is all
The working concentration of 900nM, T790M wild type and saltant type probe is 90nM, the wild type of L858R and the work of saltant type probe
Concentration is 450nM.
According on the other hand, the present invention provides the digital pcrs of human EGFR gene T790M and L858R double mutations
Detection method, this method combined using following primer and probe:
T790M upstream primer: 5 '-GCCTGCTGGGCATCTG-3 ' (SEQ ID NO.1)
T790M downstream primer: 5 '-TTTGTGTTCCCGGACATAGTC-3 ' (SEQ ID NO.2)
T790M saltant type probe: 5 '-FAM-TGAGCTGCATGATGA-MGB-NFQ-3 ' (SEQ ID NO.3)
T790M wild-type probe: 5 '-VIC-TGAGCTGCGTGATGA-MGB-NFQ-3 ' (SEQ ID NO.4)
L858R upstream primer: 5 '-CGCAGCATGTCAAGATCACAGAT-3 ' (SEQ ID NO.5)
L858R downstream primer: 5 '-CTCCTTCTGCATGGTATTCTTTCTC-3 ' (SEQ ID NO.6)
L858R saltant type probe: 5 '-FAM-AGTTTGGCCCGCCCAA-MGB-NFQ-3 ' (SEQ ID NO.7)
L858R wild-type probe: 5 '-VIC-AGTTTGGCCAGCCCAA-MGB-NFQ-3 ' (SEQ ID NO.8),
Preferably, method includes the following steps:
(1) PCR reaction system is configured:
(2) reaction system that step (1) obtains is added on digital pcr chip, then it is put into PCR instrument (for example,
ProFlexTMPCR System) in, PCR reaction is carried out,
PCR amplification program are as follows:
1) 96 DEG C, 10min；
2) 57 DEG C, 2min, 98 DEG C, 30sec, 39 circulations；
3) 57 DEG C, 2min；
4) 10 DEG C of preservations；
(3) chip after reaction is put into chip scanner and is scanned, and mutation frequency is calculated according to fluorescence signal
Rate.
Preferably, in the detection method of the digital pcr of human EGFR gene T790M and L858R double mutations of the invention,
By the different probe to T790M and primer concentration, L858R different probe and primer concentration and both of the above between not
It is adjusted with the combination of probe primer concentration ratio and the relationship of the distribution situation of signal value, the signal value distribution of the two is distinguished.
It is highly preferred that in the detection method of the digital pcr of human EGFR gene T790M and L858R double mutations of the invention, T790M
Working concentration with the upstream and downstream primer of L858R is all 900nM, and the wild type of T790M and the working concentration of saltant type probe are
The wild type of 90nM, L858R and the working concentration of saltant type probe are 450nM.
The testing principle for the chip type digital pcr that the application uses is as follows: containing 20000 nanometers on digital pcr chip
Grade micropore, mixed liquor is since the effect of hydrophobe enters in micropore and carries out PCR reaction.After PCR, containing wild type and dash forward
It will appear corresponding fluorescence signal in the micropore of modification DNA profiling, the micropore of template is not generated without fluorescence signal then.Pass through
Chip scanner is scanned these fluorescence signals, reads and analyzes, and obtains the mutation rate of T790M and L858R.
Thermo QuantStudio used in this applicationTMThe sensitivity of 3D Digital PCR detection of platform is minimum
It can reach 5/10000ths.It can expand the segment less than 100bp, facilitate the amplification and detection of tumor tissues DNA and ctDNA.
Detailed description of the invention
Fig. 1 is to show that the mutation rate of EGFR gene T790M and L858R in the embodiment of the present invention 2 are all 25% double mutation
The figure of the testing result of standard items, wherein upper figure is L858R mutation and the following figure is T790M mutation, by adjusting T790M and
The concentration and probe concentration ratio of L858R distinguishes the signal value distribution of the two.In Fig. 1, display each pair of point answer on chip one it is micro-
Hole.Wherein region 1 does not have fluorescence signal, represents no template amplification.Region 2 has FAM signal not have VIC signal, represents micropore
In the DNA profiling that is mutated containing T790M or L858R；Region 3 has VIC signal not have the representative of FAM signal to contain wild-type template；
Region 4 has VIC signal and FAM signal to represent while being mixed into wild type and saltant type template.
Fig. 2 is to show that the mutation rate of EGFR gene T790M and L858R in the embodiment of the present invention 2 are all 10% double mutation
The figure of the testing result of standard items, wherein upper figure is L858R mutation and the following figure is T790M mutation, wherein by adjusting
The concentration and probe concentration ratio of T790M and L858R distinguishes the signal value distribution of the two.In Fig. 2, display each pair of point answers chip
A upper micropore.Wherein region 1 does not have fluorescence signal, represents no template amplification.Region 2 has FAM signal not have VIC signal,
Represent the DNA profiling being mutated in micropore containing T790M or L858R；Region 3 has VIC signal not have FAM signal to represent containing wild
Pattern plate；Region 4 has VIC signal and FAM signal to represent while being mixed into wild type and saltant type template.
Fig. 3 is to show the theoretical value of the standard items of L858R difference mutation rate gradient and practical survey in substance digital pcr system
The figure of the comparison of definite value.
Fig. 4 is to show the theoretical value of the standard items of T790M difference mutation rate gradient and practical survey in substance digital pcr system
The figure of the comparison of definite value.
Specific embodiment
The following examples are intended to illustrate the invention, but is not limited to protection scope of the present invention.
Embodiment 1 is used for the primer and probe Combination Design of the dual digital pcr detection architecture of human EGFR gene.
It is used for the mankind as follows according to the mutational site T790M and L858R of human EGFR gene and adjacent to sequence design
The primer and probe of the dual digital pcr detection architecture of EGFR gene combines (SEQ ID NO.1-8):
T790M upstream primer: 5 '-GCCTGCTGGGCATCTG-3 ' (SEQ ID NO.1)
T790M downstream primer: 5 '-TTTGTGTTCCCGGACATAGTC-3 ' (SEQ ID NO.2)
T790M saltant type probe: 5 '-FAM-TGAGCTGCATGATGA-MGB-NFQ-3 ' (SEQ ID NO.3)
T790M wild-type probe: 5 '-VIC-TGAGCTGCGTGATGA-MGB-NFQ-3 ' (SEQ ID NO.4)
L858R upstream primer: 5 '-CGCAGCATGTCAAGATCACAGAT-3 ' (SEQ ID NO.5)
L858R downstream primer: 5 '-CTCCTTCTGCATGGTATTCTTTCTC-3 ' (SEQ ID NO.6)
L858R saltant type probe: 5 '-FAM-AGTTTGGCCCGCCCAA-MGB-NFQ-3 ' (SEQ ID NO.7)
L858R wild-type probe: 5 '-VIC-AGTTTGGCCAGCCCAA-MGB-NFQ-3 ' (SEQ ID NO.8)
The foundation of 2 human EGFR gene of embodiment dual digital pcr detection architecture and method
1.1 experimental material
Leucocyte genome of the used wild type gene DNA standard items from 100 normal persons in the present embodiment
DNA is mixed in equal volume.T790M and L858R mutant plasmids are bought from Origene company, the U.S., and article No. is respectively
RC400289 and RC400290.By wild type gene group, T790M mutant plasmids and L858R mutant plasmids according to certain ratio
Example carries out mixing and is respectively formed the T790M/L858R double-mutant standard items that double mutation rates are 25% and 10%.T790M and
L858R mutation rate is the specific of 25% T790M/L858R double-mutant standard items the preparation method is as follows: firstly, by glimmering
Light PCR obtains L858R plasmid stock and is diluted to 10-5It is about 10 times of 30ng/ μ L wild type gene group, T790M plasmid when gradient
Stoste is diluted to 10-5It is about 10 times of 30ng/ μ L wild type gene group when gradient；Secondly carrying out double mutation rates is 50%
The preparation of the bis- mutation standard items of T790M/L858R, in the system of 100 μ L, by 10-5L858R plasmid, 10-5T790M matter
Grain, wild type gene group and ddH2O according to volume be respectively 5 μ L, 5 μ L, 50 μ L, 40 μ L ratio mixed to get to double
The bis- mutation standard items of T790M/L858R that mutation rate is 50%；The bis- mutation of T790M/L858R for being finally 50% by double mutation rates
Standard items and wild type gene group are mixed according to the ratio of 1:1 to get the bis- mutation rate standard items of T790M/L858R to 25%,
Used ddH210 times of O dilution, it is spare.T790M and L858R mutation rate is 10% T790M/L858R double-mutant standard
Product specific the preparation method is as follows: by double mutation rates be 25% the bis- mutation standard items of T790M/L858R and wild type gene group
It mixes according to the ratio of 1:1.5 to get the bis- mutation rate standard items of T790M/L858R to 10%, is used ddH2O dilution 10
Times, it is spare.
The extraction of Whole Blood Genomic DNA
1. mixing the whole blood sample 10mL being stored in EDTA anticoagulant tube, and shift in 300 μ L to 1.5mL centrifuge tubes；
2. using whole blood genome extraction kit (the poba gene group extracts kit of Tiangeng biochemical technology Co., Ltd
DP318), wild type gene group DNA is extracted in operation to specifications；
3.NanoDrop ND-2000 is quantitative and confirms its OD260/OD280 ratio between 1.7-1.9, in order to rear
The progress of continuous experiment.
1.2 primed probe systems
By the upstream primer of L858R, downstream primer, saltant type probe and wild-type probe according to it in digital pcr system
In concentration be respectively that the ratio of 900nM, 900nM, 450nM, 450nM is mixed, be configured to 20 times of assay.Likewise,
By T790M upstream primer, downstream primer, wild-type probe and saltant type probe according to its concentration in digital pcr system point
Not Wei the ratio of 900nM, 900nM, 90nM, 90nM mixed, be configured to 20 times of assay.It can be found that the primer of T790M
Concentration is consistent with L858R, and concentration and probe concentration is the 1/5 of L858R.By adjusting the difference of probe primer concentration, make the strong of its signal
Weak generation difference, convenient for the interpretation of result and interpretation after subsequent PCR.1.3 digital pcr reaction process
(1) dual digital pcr system is prepared according to following table, wherein above-mentioned 25% or 10% T790M/L858R is bis-
Mutation rate standard items are tested as DNA profiling.
(2) according to kit specification, the above configured system is added in digital pcr chip, and uses mineral oil
Chip is covered, is sealed and ensures No leakage.
(3) chip is placed into dedicated PCR instrument, and is reacted according to following procedure.
(4) after expanding, chip is taken out from PCR instrument to room temperature, is put into progress result reading in chip scanner.
The analysis of 1.4 data results
Computer calculates mutant proportion according to fluorescence signal and manually adjusts.Its mutation rate=FAM fluorescence signal points/
(the points summation of FAM and VIC fluorescence signal) × 100%.
Since the concentration and probe concentration of T790M is far below L858R, so its FAM and VIC signal generated can also be lower than L858R,
Since the region 1 (i.e. both without FAM or the template generated without VIC signal) of the two is shared, so the signal of T790M can be more
Close to region 1, i.e., in inside.The detection of the bis- mutation standard items of the T790M/L858R that its double mutation rate is 25% and 10%
As a result as depicted in figs. 1 and 2.Its essence is exactly the concentration and probe concentration ratio by adjusting T790M and L858R, by the signal of the two
Distribution value distinguishes.
Its mutation rate and result are as shown in the table:
By upper table it can be found that under conditions of dual system, L858R and T790M it is actually detected go out mutation rate with
Its theoretical value is almost the same, which has certain feasibility.
1.5 substance digital pcr systems verify the specificity and feasibility of primed probe in dual system
(1) in order to verify the validity of dual system middle probe primer and related reagent, carry out respectively L858R and
The substance digital pcr of T790M is tested, and primer probe sequence is referring to dual digital pcr system.The primed probe concentration of L858R
Referring to dual digital pcr system, the wild type and saltant type concentration and probe concentration of T790M is all upstream and downstream primer concentration with T790M
450nM。
(2) its reaction system is as follows:
(3) under the digital pcr experiment of the standard items of L858R with T790M difference mutant proportion, testing result such as Fig. 3
And Fig. 4, theoretical value mutation rate and actually detected mutation rate all show good linear relationship, linear regression coeffficient R2?
Reach 0.99, more demonstrates the validity of above-mentioned dual digital pcr system and probe primer.
(4) measurement result of mutant proportion of L858R under the conditions of different mutation gradient standard items is as follows
(5) measurement result of mutant proportion of T790M under the conditions of different mutation gradient standard items is as follows
| Theoretical value (%) | 50 | 10 | 5 | 1 | 0.5 | 0.25 |
| Actual measured value (%) | 51.52 | 9.406 | 4.531 | 1.016 | 0.562 | 0.246 |
Although being described in detail with a general description of the specific embodiments for the present invention herein,
On the basis of the present invention, certain amendment and improvement can be carried out to it, this is aobvious and easy for those skilled in the art
See.Therefore, the modification and improvement done without departing from theon the basis of the spirit of the present invention belong to claimed model
It encloses.
SEQUENCE LISTING
<110>Bioisystech Co., Ltd, big crystal stock Thailand
<120>a kind of method and kit of detection human EGFR gene T790M and L858R mutation
<130> DI17-1314-XC37
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 16
<212> DNA
<213>artificial sequence
<220>
<223>T790M upstream primer
<400> 1
gcctgctggg catctg 16
<210> 2
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>T790M downstream primer
<400> 2
tttgtgttcc cggacatagt c 21
<210> 3
<211> 15
<212> DNA
<213>artificial sequence
<220>
<223>T790M saltant type probe
<400> 3
tgagctgcat gatga 15
<210> 4
<211> 15
<212> DNA
<213>artificial sequence
<220>
<223>T790M wild-type probe
<400> 4
tgagctgcgt gatga 15
<210> 5
<211> 23
<212> DNA
<213>artificial sequence
<220>
<223>L858R upstream primer
<400> 5
cgcagcatgt caagatcaca gat 23
<210> 6
<211> 25
<212> DNA
<213>artificial sequence
<220>
<223>L858R downstream primer
<400> 6
ctccttctgc atggtattct ttctc 25
<210> 7
<211> 16
<212> DNA
<213>artificial sequence
<220>
<223>L858R saltant type probe
<400> 7
agtttggccc gcccaa 16
<210> 8
<211> 16
<212> DNA
<213>artificial sequence
<220>
<223>L858R wild-type probe
<400> 8
agtttggcca gcccaa 16
Claims (10)
1. a kind of primer and probe group of the digital pcr detection architecture for human EGFR gene T790M and L858R double mutations
It closes, the primer and probe combination includes: for the primer and probe combination of T790M and for the primer and probe group of L858R
It closes,
Wherein, the primer and probe for T790M combines are as follows:
T790M upstream primer: 5 '-GCCTGCTGGGCATCTG-3 ' (SEQ ID NO.1)
T790M downstream primer: 5 '-TTTGTGTTCCCGGACATAGTC-3 ' (SEQ ID NO.2)
T790M saltant type probe: 5 '-FAM-TGAGCTGCATGATGA-MGB-NFQ-3 ' (SEQ ID NO.3)
T790M wild-type probe: 5 '-VIC-TGAGCTGCGTGATGA-MGB-NFQ-3 ' (SEQ ID NO.4),
Wherein, it is combined for the primer and probe of L858R are as follows:
L858R upstream primer: 5 '-CGCAGCATGTCAAGATCACAGAT-3 ' (SEQ ID NO.5)
L858R downstream primer: 5 '-CTCCTTCTGCATGGTATTCTTTCTC-3 ' (SEQ ID NO.6)
L858R saltant type probe: 5 '-FAM-AGTTTGGCCCGCCCAA-MGB-NFQ-3 ' (SEQ ID NO.7)
L858R wild-type probe: 5 '-VIC-AGTTTGGCCAGCCCAA-MGB-NFQ-3 ' (SEQ ID NO.8).
2. a kind of digital pcr detection architecture for human EGFR gene T790M and L858R double mutations, it includes rights to want
The combination of primer and probe described in asking 1.
3. digital pcr detection architecture as claimed in claim 2, wherein the concentration and probe concentration ratio of T790M and L858R is set as
Distinguish the signal value distribution of the two.
4. digital pcr detection architecture as claimed in claim 2, wherein the work of the upstream and downstream primer of T790M and L858R is dense
Degree is all 900nM, and the wild type of T790M and the working concentration of saltant type probe are 90nM, and the wild type and saltant type of L858R is visited
The working concentration of needle is 450nM.
5. digital pcr detection architecture as claimed in claim 2, which is characterized in that the digital pcr detection architecture is also wrapped
It includes: digital pcr reaction premixed liquid；DNA profiling；ddH2O。
6. a kind of digital pcr detection kit for human EGFR gene T790M and L858R double mutations, it includes rights
It is required that primer and probe described in 1 combines.
7. digital pcr detection kit as claimed in claim 6, which is characterized in that the kit further includes that digital pcr is anti-
The premixed liquid answered, for example, QuantStudioTM 3D Digital PCR Master Mix v2。
8. digital pcr detection kit as claimed in claim 6, wherein the work of the upstream and downstream primer of T790M and L858R
Concentration is all 900nM, and the working concentration of T790M wild type and saltant type probe is 90nM, and the wild type and saltant type of L858R is visited
The working concentration of needle is 450nM.
9. a kind of detection method of the digital pcr of human EGFR gene T790M and L858R double mutations, this method uses as follows
Primer and probe combination:
T790M upstream primer: 5 '-GCCTGCTGGGCATCTG-3 ' (SEQ ID NO.1)
T790M downstream primer: 5 '-TTTGTGTTCCCGGACATAGTC-3 ' (SEQ ID NO.2)
T790M saltant type probe: 5 '-FAM-TGAGCTGCATGATGA-MGB-NFQ-3 ' (SEQ ID NO.3)
T790M wild-type probe: 5 '-VIC-TGAGCTGCGTGATGA-MGB-NFQ-3 ' (SEQ ID NO.4)
L858R upstream primer: 5 '-CGCAGCATGTCAAGATCACAGAT-3 ' (SEQ ID NO.5)
L858R downstream primer: 5 '-CTCCTTCTGCATGGTATTCTTTCTC-3 ' (SEQ ID NO.6)
L858R saltant type probe: 5 '-FAM-AGTTTGGCCCGCCCAA-MGB-NFQ-3 ' (SEQ ID NO.7)
L858R wild-type probe: 5 '-VIC-AGTTTGGCCAGCCCAA-MGB-NFQ-3 ' (SEQ ID NO.8),
Preferably, method includes the following steps:
(1) PCR reaction system is configured:
(2) reaction system that step (1) obtains is added on digital pcr chip, then it is put into PCR instrument (for example,
ProFlexTMPCR System) in, PCR reaction is carried out,
PCR amplification program are as follows:
1) 96 DEG C, 10min；
2) 57 DEG C, 2min, 98 DEG C, 30sec, 39 circulations；
3) 57 DEG C, 2min；
4) 10 DEG C of preservations；
(3) chip after reaction is put into QuantStudioTMIn 3D Digital PCR Chip Loader chip scanner
It is scanned, and the frequency of mutation is calculated according to fluorescence signal.
10. the detection method of the digital pcr of human EGFR gene T790M as claimed in claim 9 and L858R double mutations,
Wherein, by different probe and primer concentration to T790M, the different probe of L858R and primer concentration and both of the above it
Between the combination of different probe primer concentration ratio and the relationship of the distribution situation of signal value adjust, by the signal value distributed area of the two
It separates；Preferably, the working concentration of the upstream and downstream primer of T790M and L858R is all 900nM, the wild type and saltant type of T790M
The working concentration of probe is 90nM, and the wild type of L858R and the working concentration of saltant type probe are 450nM.
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