CN110343747A - A kind of definite value primer and probe group correcting EGFR genetic mutation frequency - Google Patents
A kind of definite value primer and probe group correcting EGFR genetic mutation frequency Download PDFInfo
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Abstract
The invention discloses a kind of definite value primer and probe groups for correcting EGFR genetic mutation frequency, more particularly to the digital pcr probe system in 4 mutational sites of EGFR gene, pass through optimum experimental, correct parting and good linear performance are reached, by referring to quality-control product, correction coefficient is successfully effectively assessed the definite value of gene frequency and be calculated, realizes the correction to definite value experimental system.
Description
Technical field
The invention belongs to molecular biology fields, and in particular to a kind of definite value primer for correcting EGFR genetic mutation frequency
With probe groups and method.
Background technique
EGFR (Epidermal Growth Factor Receptor) is epidermal growth factor (EGF) cell Proliferation and letter
Number conduction receptor, belong to receptor tyrosine kinase family.EGFR mutation or overexpression can generally cause tumour.The overexpression of EGFR
With the transfer of tumour cell, to invade profit, poor prognosis related.Inner, the frequency of mutation of EGFR gene in non-small cell adenocarcinoma of lung (NSCLC)
It is very high, it is the hot spot mutation gene of non-small cell lung cancer, the especially non-smoking female patient of asian ancestry.For EGFR gene
Mutational site and corresponding targeted drug are also studied clearer.The common mutations site of EGFR gene occurs 18,19,
On 20 and 21 exons, wherein the mutation of 19 exons and 21 exons is most common, 90% or so be entirely mutated is accounted for,
The non-frameshift deletion mutation of 19 exons accounts for about 45%, and the L858R point mutation of 21 exons accounts for 40-45%, both are prominent
Become and is referred to as common mutations.It is in recent years with the small molecule tyrosine kinase inhibitors TKI that EGFR gene is representative target spot
One of the hot spot of NSCLC targeted therapy research, multinomial research confirm that EGFR genetic mutation is the decision of EGFR-TKI treatment curative effect
Sexual factor.Research it is also shown that TKI curative effect not only to whether have that EGFR genetic mutation is related, also have with the amount of gene mutation close
Cut relationship.In addition, the T790M somatic mutation of EGFR gene extron 20 be the secondary drug resistant main mechanism of EGFR-TKI it
One, mutation is so that Patients with Non-small-cell Lung secondary drug resistance occurs to Gefitinib and Erlotinib.Therefore, the suitable inspection of selection
Survey method filters out targeted therapy beneficiaries with important clinical meaning, and it is prominent that EGFR gene is detected especially in patients with lung cancer
Becoming the screening to targeted drug has very important directive significance.
Currently, the method for detection EGFR genetic mutation mainly has Sanger PCR sequencing PCR and high pass based on sequencing technologies to measure
ARMS-PCR (the amplification- of sequence method (Next Generation Sequencing, NGS) and based on PCR technology
Refractory mutation system, ARMS), mutation enrichment PCR and COLD-PCR.And these methods its specificity and
There is very big difference in terms of sensitivity.As generation sequencing approach (Sanger sequencing) is being applied to measurement allelic mutation Shi Suoneng
The sensitivity 20% or so of measurement；And the sensitivity of high-flux sequence method is then in 2-6% or so.Clinical labororatory is common
ARMS-PCR method detects the mutation of plasma free Tumour DNA T790M, with easy to operate, the time-consuming advantages such as few, however
Its sensitivity is still to be improved, 1% or so.Such as when the abundance that EGFR drug resistance site T790M is mutated in blood sample compares
When low, certain false negative will cause, so that a part of patient's EGFR gene medicament-resistant mutation is missed.
Digital pcr is emerging liquid biopsy method a kind of in recent years, is theoretically had compared with ARMS-PCR higher
Precision, repeatability and sensibility, and can absolute quantitation, sensitivity can achieve 0.01%, starts gradually to substitute ARMS-PCR.Number
Word PCR method occupies an important position in noninvasive cycling tumor DNA detection, has played instrument detection sensitivity and parting is accurate
Advantage.However, the sensitivity for analysis due to distinct methods is different, the inconsistent of testing result and not comparable, and difference will lead to
There are relatively big difference in nucleic acid definite value result, main cause may be probe joint efficiency, visit for enterprise product and experimental system
Needle fluorescence signal stability, primer and probe the factors such as interfere with each other and cause.Frequency of mutation definite value has in diagnostic products in vitro
It is significant, as the accepted method of nucleic acid definite value, how to accomplish the superiority and inferiority of correctly evaluation experimental system, currently without conjunction
Suitable method system.It is badly in need of the standardized methods such as the EGFR plasmid standards for quantitation with accurate mutant proportion or valued methods to examine
Comparativity between the sensitivity and specificity of various analysis methods and various methods.
Summary of the invention
The reference method that digital pcr instrument is measured as nucleic acid, plays important use in the definite value of gene mutation frequency.
However, different digital pcr instrument and reagent when detecting EGFR genetic mutation frequency there are significant difference, the present invention devises
A kind of experimental calculation method correctly evaluation appts and reagent and can calculate the frequency of mutation.In the present invention, EGFR base is corrected
Because the valued methods of the frequency of mutation are not related to the diagnosis of disease, the accuracy of digital pcr experimental system, therefore the present invention can determine whether
Method belong to non-diagnostic method.
The object of the present invention is to provide a kind of definite value experimental system of EGFR genetic mutation frequency and method, this method is relied on
It is detected in digital pcr, has given full play to digital pcr detection sensitivity and the accurate advantage of parting.The definite value tests body
System and method are different from previous EGFR genetic mutation detection, the reliability with magnitude.The experimental system devises a set of spy
Needle and primer combination, and the correction coefficient between different loci is introduced, so that different probe obtains preferably in nucleic acid quantification
Consistency.
Specifically, the present invention relates to a kind of valued methods for correcting EGFR genetic mutation frequency, comprising the following steps: 1) divide
The primer and probe of detection EGFR genetic mutation type and wild type, and the primer and probe of detection reference gene are not prepared；3)
Digital pcr is carried out using above-mentioned primer and probe, obtains EGFR genetic mutation type, EGFR gene wild type and reference gene
Copy number；4) quantitative copy number correction coefficient n is calculated, the frequency of mutation is corrected using the correction coefficient n.
Specifically, the detection EGFR genetic mutation type primed probe, selected from the detection mutational site EGFR G719S,
One of primer and probe of Ex19del, T790M and L858R is a variety of.
The primer particular sequence of described the detection mutational site EGFR G719S, Ex19del, T790M and L858R may is that
EGFR-G719S F CTTACACCCAGTGGAGAAGC SEQ ID No.1
EGFR-G719S R TGCCAGGGACCTTACCTTAT SEQ ID No.2
EGFR-Ex19del F TCTGTCATAGGGACTCTGGAT SEQ ID No.3
EGFR-Ex19del R AGCAAAGCAGAAACTCACATC SEQ ID No.4
EGFR-T790M F CATCTGCCTCACCTCCACC SEQ ID No.5
EGFR-T790M R AGCAGGTACTGGGAGCCAAT SEQ ID No.6
EGFR-L858R F AGCCAGGAACGTACTGGTGA SEQ ID No.7
EGFR-L858R R TGCCTCCTTCTGCATGGTAT SEQ ID No.8。
The wild type and saltant type probe point of described the detection mutational site EGFR G719S, Ex19del, T790M and L858R
Fluorescent marker that Shi Yong be not different, such as wild-type probe use VIC, and saltant type probe uses FAM.
Specifically, the wild type and saltant type probe in the detection mutational site EGFR may is that
EGFR-G719S WT VIC-ATCAAAGTGCTGGGCTC-MGB-NFQ SEQ ID No.13
EGFR-G719S MU FAM-ATCAAAGTGCTGAGCTC-MGB-NFQ SEQ ID No.14
EGFR-Ex19del WT VIC-TCAAGGAATTAAGAGAAGC-MGB-NFQ SEQ ID No.15
EGFR-Ex19del MU FAM-TCGCTATCAAGACATCT-MGB-NFQ SEQ ID No.16
EGFR-T790M WT VIC-ATGAGCTGCGTGATGAG-MGB-NFQ SEQ ID No.17
EGFR-T790M MU FAM-ATGAGCTGCATGATGAG-MGB-NFQ SEQ ID No.18
EGFR-L858R WT VIC-AGTTTGGCCAGCCCAA-MGB-NFQ SEQ ID No.19
EGFR-L858R MU FAM-AGTTTGGCCCGCCCAA-MGB-NFQ SEQ ID No.20。
Specifically, the detection reference gene is one or both of DCK and/or EIF5B, such as the detection DCK
And/or the primer of EIF5B may is that
DCK-Ex3 F ATGCCTGTCTCAGTCGAATAAG SEQ ID No.9
DCK-Ex3 R GCCACGTACAAGCCATTTATAC SEQ ID No.10
EIF5B-Ex1 F GCTGAGCGGAGACCAAAGT SEQ ID No.11
EIF5B-Ex1 R TCTACCTGTCTTCGCTCTTGTT SEQ ID No.12。
The probe of the detection DCK and/or EIF5B may is that
DCK-Ex3 FAM-CTCAGCTTGCCTCTC-MGBNFQ SEQ ID No.21
EIF5B-Ex1 FAM-CGGGAGACAGTGGGT-MGB-NFQ SEQ ID No.22。
Further, the quantitative copy number correction coefficient n passes through obtains as formula (1) calculates, and wherein CP reference is ginseng
According to the copy number of gene, 2 or more reference genes are such as used, then use average.Wild type is obtained respectively and saltant type is visited
The copy number correction coefficient n of needleMUAnd nWT。
Further, the frequency of mutation is corrected using the correction coefficient n, i.e., the described frequency of mutation AF passes through as public
Formula (2), which calculates, to be obtained, wherein CPMUFor saltant type probe copy number, CPwtFor wild type type probe copy number.
On the other hand, the invention further relates to a kind of definite value primer sets of EGFR genetic mutation frequency, the definite value primer sets
Primer pair including detecting EGFR genetic mutation type and wild type, and the primer pair of detection reference gene.
Specifically, the detection EGFR genetic mutation type primer pair be selected from detection the mutational site EGFR G719S, Ex19del,
The primer pair of T790M and L858R it is one or more.
Specifically, the detection EGFR genetic mutation type primer pair be selected from detection the mutational site EGFR G719S, Ex19del,
The primer pair of T790M and L858R it is one or more.
Specifically, the primer pair of described the detection mutational site EGFR G719S, Ex19del, T790M and L858R are respectively as follows:
EGFR-G719S F CTTACACCCAGTGGAGAAGC SEQ ID No.1
EGFR-G719S R TGCCAGGGACCTTACCTTAT SEQ ID No.2
EGFR-Ex19del F TCTGTCATAGGGACTCTGGAT SEQ ID No.3
EGFR-Ex19del R AGCAAAGCAGAAACTCACATC SEQ ID No.4
EGFR-T790M F CATCTGCCTCACCTCCACC SEQ ID No.5
EGFR-T790M R AGCAGGTACTGGGAGCCAAT SEQ ID No.6
EGFR-L858R F AGCCAGGAACGTACTGGTGA SEQ ID No.7
EGFR-L858R R TGCCTCCTTCTGCATGGTAT SEQ ID No.8。
Specifically, the primer pair of the detection reference gene is selected from the primer pair that can expand DCK and/or EIF5B.Into one
Step, the primer of the detection DCK and/or EIF5B is respectively as follows:
DCK-Ex3 F ATGCCTGTCTCAGTCGAATAAG SEQ ID No.9
DCK-Ex3 R GCCACGTACAAGCCATTTATAC SEQ ID No.10
EIF5B-Ex1 F GCTGAGCGGAGACCAAAGT SEQ ID No.11
EIF5B-Ex1 R TCTACCTGTCTTCGCTCTTGTT SEQ ID No.12。
On the other hand, the invention further relates to a kind of definite value probe groups of EGFR genetic mutation frequency, the definite value probe groups
Probe including detecting EGFR genetic mutation type and wild type, and the probe of detection reference gene.
Specifically, the detection EGFR genetic mutation type probe be selected from detection the mutational site EGFR G719S, Ex19del,
The probe of T790M and L858R it is one or more.Further, the detection EGFR mutational site G719S, Ex19del,
The wild type and saltant type probe of T790M and L858R uses different fluorescent markers, such as wild-type probe to use respectively
VIC, saltant type probe use FAM.
Specifically, the wild type and saltant type of described the detection mutational site EGFR G719S, Ex19del, T790M and L858R
Probe is respectively as follows:
EGFR-G719S WT VIC-ATCAAAGTGCTGGGCTC-MGB-NFQ SEQ ID No.13
EGFR-G719S MU FAM-ATCAAAGTGCTGAGCTC-MGB-NFQ SEQ ID No.14
EGFR-Ex19del WT VIC-TCAAGGAATTAAGAGAAGC-MGB-NFQ SEQ ID No.15
EGFR-Ex19del MU FAM-TCGCTATCAAGACATCT-MGB-NFQ SEQ ID No.16
EGFR-T790M WT VIC-ATGAGCTGCGTGATGAG-MGB-NFQ SEQ ID No.17
EGFR-T790M MU FAM-ATGAGCTGCATGATGAG-MGB-NFQ SEQ ID No.18
EGFR-L858R WT VIC-AGTTTGGCCAGCCCAA-MGB-NFQ SEQ ID No.19
EGFR-L858R MU FAM-AGTTTGGCCCGCCCAA-MGB-NFQ SEQ ID No.20。
Specifically, the probe of the detection reference gene is selected from the probe for being able to detect DCK and/or EIF5B.Further
, the probe of the detection DCK and/or EIF5B is respectively as follows:
DCK-Ex3 FAM-CTCAGCTTGCCTCTC-MGBNFQ SEQ ID No.21
EIF5B-Ex1 FAM-CGGGAGACAGTGGGT-MGB-NFQ SEQ ID No.22。
On the other hand, described the invention further relates to a kind of definite value primer and probe group for detecting EGFR genetic mutation frequency
Definite value primer and probe group includes the primer pair and probe of aforementioned arbitrary detection EGFR genetic mutation type and wild type, Yi Jiqian
State the primer pair and probe of arbitrary detection reference gene.
Moreover, it relates to correct the kit of EGFR genetic mutation frequency, the kit includes: 1) aforementioned
The detection EGFR genetic mutation type of meaning and the primer and probe of wild type and it is aforementioned it is arbitrary detection reference gene primer and
Probe.
The invention further relates to the definite value primer sets, definite value probe groups or definite value primer and probe groups to correct EGFR in preparation
The application of the kit of gene mutation frequency.
The invention further relates to a kind of primer pairs of reference gene and probe groups in preparation correction EGFR genetic mutation frequency
The application of kit, the reference gene are selected from least one of DCK and/or EIF5B.
Specifically, the primer pair of the detection reference gene is selected from the primer pair that can expand DCK and/or EIF5B.Into one
Step, the primer of the detection DCK and/or EIF5B is respectively as follows:
DCK-Ex3 F ATGCCTGTCTCAGTCGAATAAG SEQ ID No.9
DCK-Ex3 R GCCACGTACAAGCCATTTATAC SEQ ID No.10
EIF5B-Ex1 F GCTGAGCGGAGACCAAAGT SEQ ID No.11
EIF5B-Ex1 R TCTACCTGTCTTCGCTCTTGTT SEQ ID No.12。
Specifically, the probe of the detection reference gene is selected from the probe for being able to detect DCK and/or EIF5B.Further
, the probe of the detection DCK and/or EIF5B is respectively as follows:
DCK-Ex3 FAM-CTCAGCTTGCCTCTC-MGBNFQ SEQ ID No.21
EIF5B-Ex1 FAM-CGGGAGACAGTGGGT-MGB-NFQ SEQ ID No.22。
Technique effect and advantage of the invention is as follows:
1. the probe of design synthesis can be good at the wild type and saltant type in 4 sites of parting EGFR gene, and calculate
The different frequencies of mutation；
2. introducing correction coefficient by experimental design, effectively optimizing between different probe to frequency of mutation definite value
It corrects, realizes the superiority and inferiority of correctly evaluation experimental system.
3. by the experiment discovery of gradient frequency of mutation definite value, with the increase of copy number, the variation lines of frequency of mutation definite value
Number (CV) reduces, and in 1000 copy number, different probe definite value consistency highest, the too high or too low probe that can all increase is to fixed
The error of value specifies the set probe quantitative system optimal use range.
Detailed description of the invention
The droplet that Fig. 1 Bole digital pcr generates
The wild type and mutant plasmids of Fig. 2 synthesis
Fig. 3 plasmid PCR amplified fragments
Fig. 4 probe specific verification
The verifying of Fig. 5 probe parting
Fig. 6 probe gradient is quantitative
Fig. 7 probe quantitative curve
Tetra- kinds of probe quantitative repeatability of Fig. 8
Tetra- kinds of probe frequency of mutation definite value results of Fig. 9
Specific embodiment
The digital pcr probe system that the present invention devises 4 mutational sites of EGFR gene is reached by optimum experimental
Correct parting and preferable linear performance, by referring to quality-control product, successfully effectively assess the definite value of gene frequency
And correction coefficient is calculated, realize the correction to definite value experimental system.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.
The synthesis of 1 plasmid of embodiment, primed probe design and digital pcr system
Artificial synthesized two kinds of plasmids contain the 18th, 19,20,21 exon sequence of EGFR gene, DCK gene the 3rd respectively
Exon sequence, the 1st exon sequence of EIF5B gene.Wherein, Fusion-EGFR-Ex-WT plasmid is wild-type sequence,
It is prominent that Fusion-EGFR-Ex-MU plasmid contains p.G719S, E746-A750del (cosmic:6223), p.T790M and p.L858R
Displacement point.With Primer Express 3.0.1 and Primer Primer5.0 software design EGFR gene p.G719S,
P.E746_A750delELREA (cosmic:6225), p.T790M and p.L858R are mutated serotype specific primer probe, DCK gene and
EIF5B gene primer probe (being shown in Table 1-2).Plasmid sequence and probe primer sequence deliver the limited public affairs of Invitrogen (Shanghai) trade
Department's synthesis.
Laboratory apparatus uses U.S. Bole QX200 type droplet digital pcr instrument, and reagent is ddPCRsupermix for
Probes(#1863010).It is 20 μ L according to reagent requirement total system, three kinds of 150nM, 250nM and 400nM concentration and probe concentrations is set,
Three kinds of 55 DEG C, 57 DEG C and 60 DEG C annealing temperatures are set with optimizational primer probe experimental system, digital pcr reaction system is shown in Table 3.
Fragment amplification uses full formula gold EasyPfu DNA Ploymerase (#AP211), and primer is M13 (5 '-TGTA
AAACGACGGCCAGT-3 ' and 5 '-CAGGAAACAGCTATGACC-3 ').It is observed with Lycra inverted fluorescence microscope and generates droplet
Uniformity.
1. primer sequence information of table
2. probe sequence information of table
3. digital pcr reaction system of table
2 material of embodiment prepares and the experiment of probe qualitative, quantitative
1. instrument performance is investigated and the preparation of experimental material
By the microscopy under the microscope of the droplet after PCR, for diameter of droplets size at 110 μm or so, observed under fluorescent light is green
Color droplet is the droplet containing template DNA, it will be noted from fig. 1 that there are the droplets of small diameter in the visual field.It is in Fig. 2
Plasmid, is expanded the plasmid of synthesis by artificial synthesized wild type and mutant plasmids respectively with M13 primer, is obtained two kinds different long
The segment (Fig. 3) of degree, respectively 1483bp and 1486bp, electrophoresis run glue recycling, exclude plasmid and non-specific amplification pollution, obtain
Wild type and mutant DNA nucleic acids sample, in wild type and mutant DNA segment containing identical DCK gene and
EIF5B sequence.
2. probe specific verification
Reaction reagent is added according to 3 reaction system of table, is separately added into wild type and mutant DNA segment, verifying probe is special
The opposite sex, four kinds of typing probes uniforms are correct as the result is shown, and crossover phenomenon (Fig. 4) does not occur in fluorescence signal.By wild type and
Mutant DNA segment is mixed according to 1:1 mass ratio, does digital pcr, fluorescence signal is divided into apparent 4 quadrants as the result is shown
Region, parting are correct (Fig. 5).
3. probe linear quantitative and repeatability
Using Qubit 4.0 carry out nucleic acid quantification, respectively by wild type and mutant DNA segment be diluted to 30000 copies,
3000 copies, 1500 copies, 300 copies and 60, which copy, does digital pcr, and replication 6 times.It can see droplet fluorescence signal number
Amount successively reduces (Fig. 6), and quantitative copy number numerical value is taken log respectively10, linear fit discovery VIC and FAM signal probe all have
Preferable consistency, related coefficient are all larger than between the probe of 0.99, VIC signal that there are certain numerical fluctuations (Fig. 7).
The verifying of 3 digital pcr of embodiment and copy number correction
1. the instrument used
BIO-RAD QX200 droplet digital PCR；Superclean bench；Biohazard Safety Equipment；Gel-electrophoretic apparatus；It is solidifying
Glue imager；Nucleic acid quantification instrument；10 μ L, 100 μ L and 1000 μ L pipettors；
2. the reagent used
Bio-Rad ddPCR Supermix for Probe、Droplet Generation Oil for Probes、
DG8 Cartiridges, DG8 Gaskets, 96-well plates, Foil Seals, 0.2mL, 1mL Ep pipe.
3. environmental requirement
A) 100,000 grades of clean rooms；Work room temperature is answered constant, can control at 25 DEG C ± 3 DEG C；
4. operating procedure
Reaction solution is configured according to the experimental system provided in table 3, according to BIO-RAD QX200 digital pcr operating method
It is tested.According to the probe and primer of design, plasmid Fusion-EGFR-Ex-WT and Fusion-EGFR-Ex- are tested respectively
MU obtains copy number results.
5, interpretation of result
In wild type and mutant DNA segment contain identical DCK gene and EIF5B gene order, so, DCK and
EIF5B probe is theoretically completely the same in wild type and mutant DNA segment copy number quantitative result.It can from Fig. 7
It arriving, the quantitative values of G719S, Ex19del, T790M, L858R probe and DCK and EIF5B probe coincide substantially, but G719S,
Between the obtained VIC signal (wild type) of Ex19del, T790M, L858R probe system and FAM signal (saltant type) copy number also
It is to have a certain difference.
Using the average result that DCK and EIF5B probe obtains as the wild of G719S, Ex19del, T790M, L858R probe
The reference of type and saltant type copy number.The quantitative copy number correction coefficient (n) of each probe is obtained according to formula (1), referring to table 4.
Wherein CP reference is the copy number of reference gene, such as uses 2 or more reference genes, then uses average.Point
Not Huo get wild type and saltant type probe copy number correction coefficient nmuAnd nwt。
4. probe quantitative copy number correction coefficient of table
As can see from Figure 8, when 4 kinds of probe assay difference copy numbers, the repeatability of VIC signal and FAM signal does not have
Notable difference, average coefficient of variation (CV) is successively reduced with the increase of copy number, and in 60 copy number, CV value is maximum, is
9.8%；And in 30000 copy number, CV value is minimum, and only 0.23%, illustrate that digital pcr has preferably when measure high copy number
Repeatability.
5. the frequency of mutation corrects
The frequency of mutation that sample can be calculated according to formula (2), by wild type and mutant DNA segment according to 1:1 mass
Ratio mixing is diluted to 100000 copies, 10000 copies, 5000 copies, 1000 copies and 100 copies, the sample of different copy numbers
This does digital pcr definite value respectively.
Theoretically, the frequency of mutation that different copy number samples obtain is answered identical, is 50%.However, due to probe
The factors such as joint efficiency, fluorescence probe signal stabilization, droplet signal strength or weakness, the frequency of mutation that each probe obtains can exist certain
Difference, sample is repeated 6 times the quantitative result being averaged as probe.As shown in figure 9, the frequency of mutation as a result, it has been found that, mutation
Frequency is up to 56.1%, and minimum 50.1%, the average frequency of mutation is 52.9%, and each probe frequency of mutation CV value that is averaged is
3.2%.Illustrate the EGFR genetic mutation frequency after correcting, there is preferable consistency between each probe.And copy number is 1000
When copy number, the definite value result difference of four kinds of probes is minimum.
Based on above-mentioned, the definite value experimental system of EGFR genetic mutation frequency, it is different from previous EGFR genetic mutation detection,
Reliability with magnitude.Digital pcr method occupies an important position in noninvasive cycling tumor DNA detection, has played instrument inspection
Survey sensitivity and the accurate advantage of parting.However, how the accepted method as nucleic acid definite value, accomplish correctly evaluation experimental body
The superiority and inferiority of system, but without suitable method system.Devise the digital pcr probe in 4 mutational sites of EGFR gene first herein
System has reached correct parting and preferable linear performance, by referring to quality-control product, successfully to gene by optimum experimental
Correction coefficient is effectively assessed and be calculated to the definite value of frequency, realizes the correction to definite value experimental system.
It is not for limiting claim, any this field skill although the present invention is disclosed as above with preferred embodiment
Art personnel without departing from the inventive concept of the premise, can make several possible variations and modification, therefore of the invention
Protection scope should be quasi- with range that the claims in the present invention are defined.
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
tcgctatcaa gacatct 17
<210> 17
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
atgagctgcg tgatgag 17
<210> 18
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
atgagctgca tgatgag 17
<210> 19
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
agtttggcca gcccaa 16
<210> 20
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
agtttggccc gcccaa 16
<210> 21
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
ctcagcttgc ctctc 15
<210> 22
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
cgggagacag tgggt 15
Claims (18)
1. a kind of definite value primer sets of EGFR genetic mutation frequency, which is characterized in that the definite value primer sets include detection EGFR
The primer pair of genic mutation type and wild type, and the primer pair of detection reference gene.
2. definite value primer sets according to claim 1, which is characterized in that the primer pair of the detection reference gene is selected from and can expand
Increase the primer pair of DCK and/or EIF5B.
3. definite value primer sets according to claim 1 or 2, which is characterized in that the primer difference of the detection DCK and/or EIF5B
Are as follows:
DCK-Ex3 F ATGCCTGTCTCAGTCGAATAAG SEQ ID No.9
DCK-Ex3 R GCCACGTACAAGCCATTTATAC SEQ ID No.10
EIF5B-Ex1 F GCTGAGCGGAGACCAAAGT SEQ ID No.11
EIF5B-Ex1 R TCTACCTGTCTTCGCTCTTGTT SEQ ID No.12。
4. definite value primer sets according to claim 1, which is characterized in that the detection EGFR genetic mutation type primer pair is selected from inspection
Survey the one or more of the primer pair of the mutational site EGFR G719S, Ex19del, T790M and L858R.
5. according to claim 1 or 4 definite value primer sets, which is characterized in that specifically, the mutational site detection EGFR
The primer pair of G719S, Ex19del, T790M and L858R are respectively as follows:
EGFR-G719S F CTTACACCCAGTGGAGAAGC SEQ ID No.1
EGFR-G719S R TGCCAGGGACCTTACCTTAT SEQ ID No.2
EGFR-Ex19del F TCTGTCATAGGGACTCTGGAT SEQ ID No.3
EGFR-Ex19del R AGCAAAGCAGAAACTCACATC SEQ ID No.4
EGFR-T790M F CATCTGCCTCACCTCCACC SEQ ID No.5
EGFR-T790M R AGCAGGTACTGGGAGCCAAT SEQ ID No.6
EGFR-L858R F AGCCAGGAACGTACTGGTGA SEQ ID No.7
EGFR-L858R R TGCCTCCTTCTGCATGGTAT SEQ ID No.8。
6. a kind of definite value probe groups of EGFR genetic mutation frequency, the definite value probe groups include detection EGFR genetic mutation type and
The probe of wild type, and the probe of detection reference gene.
7. definite value probe groups according to claim 6, which is characterized in that the probe of the detection reference gene, which is selected from, to be able to detect
The probe of DCK and/or EIF5B.
8. the definite value probe groups of according to claim 6 or 7, which is characterized in that the probe difference of the detection DCK and/or EIF5B
Are as follows:
DCK-Ex3 FAM-CTCAGCTTGCCTCTC-MGBNFQ SEQ ID No.21
EIF5B-Ex1 FAM-CGGGAGACAGTGGGT-MGB-NFQ SEQ ID No.22。
9. definite value probe groups according to claim 6, which is characterized in that the detection EGFR genetic mutation type probe is selected from detection
The probe of the mutational site EGFR G719S, Ex19del, T790M and L858R it is one or more.
10. definite value probe groups according to claim 9, which is characterized in that the detection EGFR mutational site G719S,
The wild type and saltant type probe of Ex19del, T790M and L858R use different fluorescent markers respectively.
11. definite value probe groups according to claim 10, which is characterized in that the wild-type probe uses VIC, the mutation
Type probe uses FAM.
12. definite value probe groups according to claim 9 or 10, which is characterized in that the detection EGFR mutational site G719S,
The wild type and saltant type probe of Ex19del, T790M and L858R are respectively as follows:
EGFR-G719S WT VIC-ATCAAAGTGCTGGGCTC-MGB-NFQ SEQ ID No.13
EGFR-G719S MU FAM-ATCAAAGTGCTGAGCTC-MGB-NFQ SEQ ID No.14
EGFR-Ex19del WT VIC-TCAAGGAATTAAGAGAAGC-MGB-NFQ SEQ ID No.15
EGFR-Ex19del MU FAM-TCGCTATCAAGACATCT-MGB-NFQ SEQ ID No.16
EGFR-T790M WT VIC-ATGAGCTGCGTGATGAG-MGB-NFQ SEQ ID No.17
EGFR-T790M MU FAM-ATGAGCTGCATGATGAG-MGB-NFQ SEQ ID No.18
EGFR-L858R WT VIC-AGTTTGGCCAGCCCAA-MGB-NFQ SEQ ID No.19
EGFR-L858R MU FAM-AGTTTGGCCCGCCCAA-MGB-NFQ SEQ ID No.20。
13. a kind of definite value primer and probe group of EGFR genetic mutation frequency, which is characterized in that the definite value primer and probe group
The definite value probe groups of definite value probe groups and/or claim 6-12 any one selected from claim 1-5 any one.
14. a kind of kit for correcting EGFR genetic mutation frequency, which is characterized in that the kit includes claim 1-5
The definite value probe groups of any one and/or the definite value probe groups of claim 6-12 any one.
15. a kind of definite value primer sets of claim 1-5 any one, the definite value probe groups of claim 6-12 any one,
Application of the definite value primer and probe group of claim 13 in the kit of preparation correction EGFR genetic mutation frequency.
16. the primer pair and probe groups of a kind of reference gene correct the application of the kit of EGFR genetic mutation frequency in preparation,
It is characterized in that, the reference gene is selected from least one of DCK and/or EIF5B.
17. application according to claim 16, which is characterized in that the reference gene DCK and/or EIF5B primer pair are respectively as follows:
DCK-Ex3 F ATGCCTGTCTCAGTCGAATAAG SEQ ID No.9
DCK-Ex3 R GCCACGTACAAGCCATTTATAC SEQ ID No.10
EIF5B-Ex1 F GCTGAGCGGAGACCAAAGT SEQ ID No.11
EIF5B-Ex1 R TCTACCTGTCTTCGCTCTTGTT SEQ ID No.12。
18. 6 or 17 application according to claim 1, which is characterized in that the probe of the reference gene DCK and/or EIF5B point
Not are as follows:
DCK-Ex3 FAM-CTCAGCTTGCCTCTC-MGBNFQ SEQ ID No.21
EIF5B-Ex1 FAM-CGGGAGACAGTGGGT-MGB-NFQ SEQ ID No.22。
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| WO2016167317A1 (en) * | 2015-04-14 | 2016-10-20 | 凸版印刷株式会社 | Method for detecting genetic mutation |
| CN106520931A (en) * | 2016-10-17 | 2017-03-22 | 上海赛安生物医药科技有限公司 | EGFR gene mutation detection primer probe and kit thereof |
| CN108611412A (en) * | 2018-03-28 | 2018-10-02 | 无锡市申瑞生物制品有限公司 | A kind of primer combination of probe of EGFR genetic mutation detection and its application |
| CN108642154A (en) * | 2018-06-01 | 2018-10-12 | 领航基因科技（杭州）有限公司 | The primer combination of probe and kit in a kind of detection EGFR mutational sites and its application |
| CN108949990A (en) * | 2018-08-08 | 2018-12-07 | 中山大学达安基因股份有限公司 | A kind of kit and method detecting EGFR genetic mutation |
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| WO2016167317A1 (en) * | 2015-04-14 | 2016-10-20 | 凸版印刷株式会社 | Method for detecting genetic mutation |
| CN105624309A (en) * | 2016-02-23 | 2016-06-01 | 深圳华大基因研究院 | Primer, probe and kit for detecting EGFR and/or K-ras genetic mutation |
| CN106520931A (en) * | 2016-10-17 | 2017-03-22 | 上海赛安生物医药科技有限公司 | EGFR gene mutation detection primer probe and kit thereof |
| CN108611412A (en) * | 2018-03-28 | 2018-10-02 | 无锡市申瑞生物制品有限公司 | A kind of primer combination of probe of EGFR genetic mutation detection and its application |
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