CN107365868A - BRAF and EGFR genetic mutation detection plasmid standards for quantitation and preparation method thereof and valued methods - Google Patents
BRAF and EGFR genetic mutation detection plasmid standards for quantitation and preparation method thereof and valued methods Download PDFInfo
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Abstract
The invention discloses a kind of BRAF gene and EGFR abrupt climatic change plasmid standards for quantitation and preparation method thereof and valued methods, this method is built containing BRAF gene V600E respectively, EGFR gene T790M, L858R and the plasmid in deletion mutation E746_A750del mutational sites, enter performing PCR amplification by template of plasmid, PCR primer is obtained after Sanger sequence verifications, weighed by balance with the wild type human genomic DNA by ultrasonic wave fragmentation and mixed according to a certain percentage, BRAF gene V600E mutation and EGFR gene T790M can be obtained, the plasmid standards for quantitation of L858R and deletion mutation E746_A750del mutation.The invention provides the preparation method of plasmid standards for quantitation, using the wild type human genomic DNA of fragmentation as background dna, the background of actually detected dissociative DNA sample can be preferably simulated.The valued methods precision of this plasmid standards for quantitation is good, and the degree of accuracy is high, and independent of DNA standard items, measurement result can trace to the source to international base unit (quality), so as to ensure the reliable of measurement result and can trace to the source.
Description
Technical field
The present invention relates to field of gene detection, more particularly to a kind of BRAF and EGFR genetic mutation plasmid standards for quantitation.
Background technology
BRAF is one of most important proto-oncogene of the mankind, and BRAF mutation occur for about 8% human tumor.BRAF is big absolutely
Fractional mutations form is mutated for BRAF V600E, takes place mostly in melanoma, colon cancer and thyroid cancer.The mutation causes
Downstream MEK-ERK signal path sustained activations, the growing multiplication and invasion and attack transfer to tumour are most important, are melanomas etc.
One of useful effect target of V600E mutated tumors.2011, first BRAFV600E targeted inhibitions agent Wei Luofeini was by FDA batches
Quasi- listing, for treating the advanced melanoma patient of BRAFV600E mutation, effectively extend patient's progression free survival phase and total
Life cycle, achieve breakthrough therapeutic effect, and the target therapeutic agent typically based on gene diagnosis selection medication.Cause
This detection to BRAF gene mutation type, there is very important directive significance to selection target therapeutic agent.
EGFR (Epidermal Growth Factor Receptor) is epidermal growth factor (EGF) cell propagation and letter
Number conduction acceptor.EGFR, which is mutated or is overexpressed, can typically trigger tumour.EGFR overexpression and the transfer of tumour cell, invade
Profit, poor prognosis are relevant.In non-small cell adenocarcinoma of lung, the frequency of mutation of EGFR gene is very high, and especially asian ancestry are non-smoking
Female patient.Mutational site and corresponding targeted drug for EGFR gene are also studied clearer.EGFR gene
Common mutations site occurs on 18,19,20 and 21 exons, wherein the non-frameshift deletion mutation of 19 exons accounts for
45%, the L858R point mutation of 21 exons accounts for 40-45%, and both mutation are referred to as common mutations.EGFR gene has medicine
Certain targeted drug can be used after sensitizing mutation, that is, mutation, also there is resistance site, that is, to certain targeted drug after being mutated
Resistance.Therefore screening of the EGFR genetic mutation to targeted drug is detected in patients with lung cancer has very important directive significance.
At present, detecting BRAF and the method for EGFR genetic mutation mainly has Sanger PCR sequencing PCRs and height based on sequencing technologies
Flux PCR sequencing PCR (Next Generation Sequencing, NGS), and the ARMS-PCR of PCR-based technology
(amplification-refractory mutation system, ARMS), mutation enrichment PCR and COLD-PCR.And these
Method has very big difference in terms of its specificity and sensitivity.Such as generation sequence measurement (Sanger sequencings) is applied to measure
The sensitivity 20% or so that can be determined during the gene mutation of position；And the sensitivity of high-flux sequence method is then in 2-6% or so.
The sensitivity for analysis of ARMS-PCR methods is 1%, and the sensitivity for being mutated enrichment PCR and COLD-PCR is higher, can reach
0.1%.Because the sensitivity for analysis of distinct methods is different, the inconsistent of testing result and not comparable can be caused.Therefore, it is badly in need of tool
There is BRAF the and EGFR plasmid standards for quantitation of accurate mutant proportion, to examine the sensitivity and specificity of various analysis methods, and
Comparativity between various methods.
The country there is no the plasmid standards for quantitation for people BRAF and EGFR gene detection at present.
The content of the invention
It is an object of the invention to provide a kind of BRAF and EGFR genetic mutation plasmid standards for quantitation and preparation method thereof and answer
With.The BRAF gene mutation plasmid standards for quantitation degree of accuracy that this method obtains is high, with respect to expanded uncertainty 2%~3%.
The present invention establishes the preparation method of BRAF and EGFR genetic mutation plasmid standards for quantitation first, has reached foregoing invention
Purpose.
The technical scheme is that：
(1) plasmid containing BRAF and EGFR genetic mutation is built, is expanded and is contained using regular-PCR as template using plasmid
There is the genetic fragment in targeted mutagenesis site, the purity in the mutational site of PCR primer is sequenced by Sanger.
(2) wild type human genomic DNA is extracted, carries out fragmentation.By the wild of the PCR primer of targeted mutagenesis and fragmentation
Type human gene group DNA mixes according to a certain percentage.
(3) add in yeast total serum IgE to the wild type human genomic DNA mixed solution of PCR primer and fragmentation.
Wherein, the constructed plasmid containing BRAF and EGFR genetic mutation site is to contain BRAF gene mutation type respectively
V600E, EGFR genetic mutation type T790M, L858R and E746_A750del 4 plasmids.
Its specific method is：
Before mixing according to a certain percentage, first by the PCR primer of targeted mutagenesis and the wild type human genome of fragmentation
DNA is diluted to identical copy Particle density.
By the wild type human genomic DNA of the PCR primer containing targeted mutagenesis and ultrasonic tear reason fragmentation according to certain ratio
Example mixing, can be mixed using balance weighing method.
Described fragmentation refers to make its fragmentation using ultrasonication, and preferable experiment condition is：Gathered using sound wave
Burnt sonicator processing, intensity：5, the time：6min, applied sample amount are 100 μ L, DNA concentration：10-50ng/ μ L, work follow
Ring：10%, explosion/circulation：200, temperature：4℃；
In some embodiments, the wild type gene group DNA of PCR primer and fragmentation is diluted to respectively before combination
5000±280copies/μL。
Targeted mutagenesis frequency values calculation formula is as follows in the plasmid standards for quantitation of the present invention：
Wherein mMUFor the mass value of the PCR primer in the site containing targeted mutagenesis, cMUFor the PCR primer in the site containing targeted mutagenesis
Copy Particle density, mWTFor wild type gene group DNA mass value, cWTFor wild type gene group DNA copy Particle density.
In a further preferred embodiment, pressed by the genomic DNA of the PCR primer containing targeted mutagenesis and fragmentation
It is X according to mixed proportion:Y, wherein X are that 0.01~5, Y is 5~9.99, and X+Y=10 ratio (mass ratio) mixes.
Yeast total rna concentration is 300~400ng/ μ L in a further preferred embodiment.
BRAF and EGFR genetic mutation the detection plasmid standards for quantitation of the present invention carries out definite value using gravimetric method, without external standard,
Quantitative result cocoa is traced to the source to international base unit (quality), so as to ensure the reliable of standard items value and can trace to the source.The present invention
BRAF and EGFR genetic mutation detection plasmid standards for quantitation can be as quantitative standard.The BRAF gene mutation detection of the present invention
Plasmid standards for quantitation includes common BRAF V600E mutational sites, EGFR gene T790M, L858R and E746_A750del mutation
Site, available for the assessment and quality control of BRAF and EGFR genetic mutation detection method reliability and dosing accuracy, improve
Efficiency simultaneously reduces detection Quality Control cost.
Specifically, the present invention carries out the following studies work：
1. the condition of pair ultrasonication wild type human gene DNA is optimized
Sample process instrument is focused on using sound wave, processing intensity (intensity), processing time etc. are optimized.It is excellent
Changing result and see Fig. 9, it is 5 that wherein Sample 1, Sample 2, Sample 3, Sample 4 and Sample 5, which handle intensity, when
Between be respectively 3min, 4min, 5min, 6min and 8min；Sample 6, Sample 7, Sample 8, Sample 9 handle intensity
It is 4, processing time is respectively 4min, 6min, 8min and 16min.As seen from the figure, processing time 6min, processing intensity are 5
When, resulting genomic DNA fragment distribution is 50~320bp, and main peak is close with cfDNA fragment lengths in 165bp.Cause
This treatment conditions finally determined is intensity：5；Time 6min；The μ L of applied sample amount 100；Duty Cycle：10%, Cycle per
Burst：200, temperature：4℃.DNA concentration scope：10-50ng/μl.
2. pair mutant PCR products and wild type gene group DNA purity are confirmed
The homozygosity in the BRAF and EGFR mutational sites of pcr amplification product is carried out simultaneously using Sanger sequence measurements
Confirm, as a result find PCR primer by being determined as homozygous mutation (being shown in Table 1).To the pure of PCR primer and wild type gene group DNA
Degree is determined, and the results are shown in Table 2-6.
The result of the table 1 using Sanger PCR sequencing PCRs to PCR primer saltant type
The purity and concentration of table 2.BRAF V600E PCR primers
The wild type gene group DNA of table 3. purity and concentration
The purity and concentration of table 4.L858R PCR primers
The purity and concentration of table 5.E746_A750delPCR products
The purity and concentration of table 6.T790MPCR products
3. demonstrating gravimetric method prepares the uniformity that BRAF V600E results are determined with digital pcr method
By the wild type gene group DNA of PCR primer and the fragmentation of V600E mutation according to it is a certain amount of mix it is as follows
Table.Different mutation gradients are determined using digital pcr method.It is respectively by what the S1 to S4 of gravimetric method configuration was mutated abundance
67.12%th, 25.00%, 7.56%, 0.69%, the mutation abundance that S1 to S4 is obtained after ddPCR is determined is respectively
69.38%th, 24.84%, 7.23%, 0.60%.After measure, ddPCR is tied the sample of 4 different gradients with gravimetric method proportioning
Uniformity between fruit is 1.04, sees Figure 11.Hence it is demonstrated that the value of the V600E difference frequencies of mutation based on gravimetric method configuration can
To obtain the reproduction of digital pcr method.
The configuration of the different mutation abundance of table 7. and digital pcr method validation result
4. demonstrating gravimetric method prepares the uniformity that EGFR L858R results are determined with digital pcr method
The plasmid being mutated containing L858R and wild type LCL8 are mixed such as following table according to a certain percentage.Using numeral
PCR method determines 3 different mutation gradients.By gravimetric method configuration S1 to S3 mutation abundance be respectively 15.05%,
2.00%th, 0.20%, the mutation abundance that S1 to S3 is obtained after ddPCR is determined is respectively 15.01%, 2.02%, 0.19%.
For the sample of different gradients after measure, the uniformity between ddPCR and gravimetric method proportioning result is 0.990, sees Figure 12.Therefore
The reproduction of digital pcr method can be obtained by proving the value of the L858R difference frequencies of mutation based on gravimetric method configuration.
The configuration of the different mutation abundance of table 8 and digital pcr method validation result
5. demonstrating gravimetric method prepares the uniformity that EGFR T790M results are determined with digital pcr method
The plasmid being mutated containing T790M and wild type LCL8 are mixed such as following table according to a certain percentage.Using numeral
PCR method determines 3 different mutation gradients.By gravimetric method configuration S1 to S3 mutation abundance be respectively 25.51%,
4.56%th, 0.92%, the mutation abundance that S1 to S3 is obtained after ddPCR is determined is respectively 26.92%, 4.28%, 0.86%.
For the sample of different gradients after measure, the uniformity between ddPCR and gravimetric method proportioning result is 1.06, sees Figure 13.Therefore
The reproduction of digital pcr method can be obtained by proving the value of the T790M difference frequencies of mutation based on gravimetric method configuration.
The configuration of the different mutation abundance of table 9 and digital pcr method validation result
Compared with the existing technology, its innovative point and advantage are the present invention：
1st, the present invention carries out accurate quantification using gravimetric method to BRAF and EGFR genetic mutation standard items first, uses simultaneously
Digital pcr method is verified to value, it was demonstrated that its applicability.
2nd, the preparation method of BRAF and EGFR genetic mutation plasmid standards for quantitation has been started, have found suitable ultrasonic wave segment
Change the condition of genomic DNA.
3rd, weight Par value precision is good：Uncertainty is better than 3%.
4th, the method degree of accuracy is high：Adopt to weigh with scale and quantified, independent of DNA standard items, therefore measurement result can
Trace to the source to international base unit, so as to ensure the reliable of measurement result and can trace to the source.
5th, the BRAF and the wild type gene of EGFR genetic mutation plasmid standards for quantitation, wherein fragmentation prepared using this method
Dissociative DNA in group DNA simulation human bloods, and the PCR primer containing targeted mutagenesis represents Circulating tumor DNA, therefore the present invention
Standard items preferably simulate the dissociative DNA sample clinically detected.
6th, it is lower than art methods cost.
Explanation and specific embodiment are to BRAF of the present invention and the quantitative mark of EGFR genetic mutation below in conjunction with the accompanying drawings
Quasi- product and its preparation method and application are described further.
Brief description of the drawings
Fig. 1 is the result of the Sanger PCR sequencing PCRs to BRAF V600E-PCR.
Fig. 2 is the result of the Sanger PCR sequencing PCRs to BRAF V600 wild types.
Fig. 3 is the result of the Sanger PCR sequencing PCRs to EGFR L858R-PCR.
Fig. 4 is the result of the Sanger PCR sequencing PCRs to EGFR L858 wild types.
Fig. 5 is the result of the Sanger PCR sequencing PCRs to EGFR E746_A750del-PCR.
Fig. 6 is the result of the Sanger PCR sequencing PCRs to the extron wild types of EGFR 19.
Fig. 7 is the result of the Sanger PCR sequencing PCRs to EGFR T790M-PCR.
Fig. 8 is the result of the Sanger PCR sequencing PCRs to EGFR T790 wild types.
Fig. 9 is the wild type gene group DNA condition optimizing figures after ultrasonication；
Figure 10 be ultrasonication after wild type gene group DNA result figure；
Figure 11 is gravimetric method preparation and the uniformity of digital pcr method BRAF V600E results；
Figure 12 is that gravimetric method prepares the uniformity that L858R results are determined with digital pcr method；
Figure 13 is that gravimetric method prepares the uniformity that T790M results are determined with digital pcr method；
Figure 14 is that digital pcr determines BRAF mutated genes copy number two dimension scatter diagrams；
Figure 15 is that digital pcr determines BRAF wild type gene copy number two dimension scatter diagrams；
Figure 16 is the gravimetric method and digital pcr method definite value results contrast of BRAF1 plasmid standards for quantitation；
Figure 17 is the gravimetric method and digital pcr method definite value results contrast of BRAF2 plasmid standards for quantitation；
Figure 18 is the gravimetric method and digital pcr method definite value results contrast of BRAF3 plasmid standards for quantitation；
Figure 19 is the gravimetric method and digital pcr method definite value results contrast of L858R plasmid standards for quantitation.
Embodiment
Embodiment 1
The preparation of BRAF gene V600E mutation plasmid standards for quantitation and definite value
First, materials and methods：
1. cell line and culture medium
HT29 cell lines are purchased from ATCC, and required culture medium and condition of culture are carried out with reference to ATCC specifications.BRAF is wild
Raw type cell line PG-LCL8 provides for Fudan University, culture medium 1640+10%FBS, 5%CO2, 37 degree of cultures.
2.DNA is extracted and PCR amplifications
Health is used to extract the genomic DNA of cell line for the Genomic Purification Kit of ShiJi Co., Ltd, with containing targeted mutagenesis
Cell line genomic DNA enter performing PCR amplification for template, obtain the PCR primer of the targeted mutagenesis containing V600E.PCR amplification system 20
μ L, comprising concentration is 5uM 2 μ L, DNA masterplate of upstream and downstream primer 2 μ L, 2 × the μ L of PCR premixed liquids 10, the μ L of sterilized water 6.Amplification
Condition is：95 degree, 10min, 40 circulations include 95 degree of 15s, 61 degree, 30s, 72 degree 1min.
3. plasmid construction and sequence verification
PCR primer is attached by T4 ligases, competent escherichia coli cell is then converted, is sieved by blue hickie
Choosing, picking hickie carry out Liquid Culture, extract DNA, are sequenced using ABI 3500.
4.PCR products and wild type gene group purity test
Using the positive plasmid of sequence verification as template, enter performing PCR amplification, purified kit is pure again for obtained PCR primer
Change, using the micro ultraviolet specrophotometers of NanoDrop2000 determine PCR primer and wild type gene group in 260nm, 280nm and
Light absorption value at 230nm, Blank is carried out by the use of TE as blank, then takes 1 μ L samples to be measured.
5. ultrasonication
Sonicator (CovarisS2) is focused on using sound wave, treatment conditions are intensity：5；Time 6min；Applied sample amount
100μL；Duty Cycle：10%, Cycle per Burst：200, temperature：4℃.DNA concentration scope：30-40ng/μL.
6. digital pcr method measure BRAF gene copy Particle density
The copy Particle density of BRAF gene in PCR primer and wild type gene group DNA is determined using digital pcr method, is expanded
Increasing system such as table 10.The μ L of PCR reaction solutions 20 containing DNA profiling prepared are added to droplet first card mark occurs
In the hole of " Sample ", then 60 μ L droplets generation oil is added into the hole of mark " Oil ", be then placed in drop generator and generate
Droplet.It is transferred to after droplet generation in 96 orifice plates, the shrouding film of 96 orifice plates is sealed with heating instrument, is placed on regular-PCR instrument
Expanded.Amplification condition is：95 degree, 10min；40 circulations include 95 degree of 20s, 61 degree, 30s；98 degree of 10min.
PCR amplifications carry out the reading and data analysis of droplet after terminating.Point of data is carried out using QuantaSoft softwares
Analysis is handled.
Table 10.BRAF gene digital pcr amplification systems
7. gravimetric method configures the plasmid standards for quantitation of V600E saltant types
Two mutation frequencies are prepared according to the mass value of table 11 using high-precision 6 assay balances (XP56) by calibration
The plasmid standards for quantitation of rate.
Table 11. is with tabulation
8. digital pcr method validation gravimetric method preparation value
The plasmid standards for quantitation for the different mutation level that gravimetric method is prepared, is surveyed according to the operating procedure of digital pcr method
The frequency of mutation for determining the V600E saltant types in plasmid standards for quantitation calculates according to formula 1.
2nd, experimental result
1. ultrasonication wild type gene group DNA result
Result after wild type gene group DNA is ultrasonically treated is shown in Figure 10.It can be seen that after being ultrasonically treated, institute
The small fragment that the length range that some genomic DNAs have all been broken into using main peak as 165bp is 50~320bp or so, with cfDNA
Fragment length is suitable.
2.PCR is expanded and Purity result
The PCR primer genotype Purity result of V600E saltant types is shown in Fig. 1.It is right in PCR primer from Fig. 1 results
It is homozygous mutation to answer V600E saltant types, and wild type DNA does not contain any mutation.This preparation to plasmid standards for quantitation is extremely important.
In addition DNA Purities the results are shown in Table 2.According to the OD260/OD280 of measurement result PCR primer and wild type gene group 1.7
Between~1.9, OD260/OD230 is more than 2.0, therefore genotype and DNA purity are satisfied by requiring.
3. digital pcr measure BRAF gene copy Particle density
According to digital pcr to PCR primer and the genome measurement result (see Figure 14 and Figure 15) of fragmentation, to PCR primer
The positive droplet of wild type i.e. VIC marks is not detected, and does not detect FAM then for fragmentation genomic samples and marks
Positive droplet, shows to be free of saltant type in wild-type fragment genomic samples, according to respective positive droplet number, counts respectively
The copy number that calculation obtains BRAF gene in PCR primer and wild type gene group DNA is respectively 4404cp/ μ L and 4405cp/ μ L.
4. weight Par value
The saltant type and wild type gene copy Particle density determined according to the quality of weighing and digital pcr, determines preparation
Measure standard items and carry out definite value.In three horizontal plasmid standards for quantitation, the frequency of mutation corresponding to V600E saltant types be 10.7%,
1.1%th, 0.5%.
5. digital pcr determines the frequency of mutation
In order to verify whether gravimetric method preparation value can use other method to reappear, the plasmid standards for quantitation horizontal to 3 is adopted
With the frequency of mutation of digital pcr method measure V600E saltant types, 3 repetitions of each horizontal measure, measurement result such as following table.
The digital pcr measurement result of table 12
The direct uniformity of result is prepared to digital pcr measurement result with gravimetric method to analyze, such as Figure 16-18.Wherein
Round dot represents gravimetric method preparation value, triangular representation digital pcr result.Two methods measurement result uniformity is in its uncertainty
In the range of very well, but the precision of gravimetric method is better than digital pcr method, shows the use weight Par value established in the present invention
BRAF gene mutation detects plasmid standards for quantitation, and value is accurate, and applicability is good.Available for digital pcr or other method checking and
Performance Evaluation.
Embodiment 2
The preparation of EGFR gene L858R mutation plasmid standards for quantitation and definite value
First, materials and methods：
1. cell line and culture medium
The cell line of the mutation containing L858R is purchased from ATCC, and required culture medium and condition of culture enter with reference to ATCC specifications
OK.Wild-type cell system PG-LCL8 provides for Fudan University, culture medium 1640+10%FBS, 5%CO2, 37 degree of cultures.
2.DNA is extracted and PCR amplifications
Health is used to extract the genomic DNA of cell line for the Genomic Purification Kit of ShiJi Co., Ltd, with containing targeted mutagenesis
Cell line genomic DNA enter performing PCR amplification for template, obtain the PCR primer of the targeted mutagenesis containing L858R.PCR amplification system 20
μ L, comprising concentration is 5uM 2 μ L, DNA masterplate of upstream and downstream primer 2 μ L, 2 × the μ L of PCR premixed liquids 10, the μ L of sterilized water 6.Amplification
Condition is：95 degree, 10min, 40 circulations include 95 degree of 15s, 61 degree, 30s, 72 degree 1min.
3. plasmid construction and sequence verification
PCR primer is attached by T4 ligases, competent escherichia coli cell is then converted, is sieved by blue hickie
Choosing, picking hickie carry out Liquid Culture, extract DNA, are sequenced using ABI 3500.
4.PCR products and wild type gene group purity test
Using the positive plasmid of sequence verification as template, enter performing PCR amplification, purified kit is pure again for obtained PCR primer
Change, using the micro ultraviolet specrophotometers of NanoDrop2000 determine PCR primer and wild type gene group in 260nm, 280nm and
Light absorption value at 230nm, Blank is carried out by the use of TE as blank, then takes 1 μ L samples to be measured.
5. ultrasonication
Sonicator (CovarisS2) is focused on using sound wave, treatment conditions are intensity：5；Time 6min；Applied sample amount
100μL；Duty Cycle：10%, Cycle per Burst：200, temperature：4℃.DNA concentration scope：30-40ng/μL.
6. digital pcr method determines L858R gene copy Particle densities
The copy Particle density of EGFR gene in PCR primer and wild type gene group DNA is determined using digital pcr method, is expanded
Increasing system such as table 13.Experimentation determines BRAF gene copy number with above-mentioned digital pcr.
Table 13.EGFR gene digital pcr amplification systems
7. gravimetric method configures the plasmid standards for quantitation of L858R saltant types
Two mutation frequencies are prepared according to the mass value of table 14 using high-precision 6 assay balances (XP56) by calibration
The plasmid standards for quantitation of rate.
Table 14. is with tabulation
8. digital pcr method validation gravimetric method preparation value
The plasmid standards for quantitation that gravimetric method is prepared, according in the operating procedure measure plasmid standards for quantitation of digital pcr method
The frequencies of mutation of EGFR-L858R saltant types calculated according to formula 2.
2nd, experimental result
1. ultrasonication wild type gene group DNA result
Result after wild type gene group DNA is ultrasonically treated is shown in Fig. 4.It can be seen that after being ultrasonically treated, own
The length range that has all been broken into using main peak as 165bp of genomic DNA be 50~320bp or so small fragment, with cfDNA pieces
Segment length is suitable.
2.PCR is expanded and Purity result
The PCR primer genotype Purity result of L858R saltant types is shown in Fig. 3.It is right in PCR primer from Fig. 3 results
It is homozygous mutation to answer L858R saltant types, and wild type DNA does not contain any mutation.This preparation to plasmid standards for quantitation is extremely important.
In addition DNA Purities the results are shown in Table 4.According to the OD260/OD280 of measurement result PCR primer and wild type gene group 1.7
Between~1.9, OD260/OD230 is more than 2.0, therefore genotype and DNA purity are satisfied by requiring.
3. digital pcr measure EGFR gene copy Particle density
According to digital pcr to PCR primer and the genome measurement result of fragmentation, PCR primer is not detected wild
Type is the positive droplet of VIC marks, and does not detect the positive droplet of FAM marks then for fragmentation genomic samples, table
Saltant type is free of in bright wild-type fragment genomic samples, according to respective positive droplet number, PCR primer is calculated respectively
With the copy number of EGFR gene L858R wild types and saltant type in wild type gene group DNA be respectively 5103cp/ μ L and
7935cp/μL。
4. weight Par value
The saltant type and wild type gene copy Particle density determined according to the quality of weighing and digital pcr, determines preparation
Measure standard items and carry out definite value.The frequency of mutation corresponding to L858R saltant types is 5.03%.
5. digital pcr determines the frequency of mutation
In order to verify whether gravimetric method preparation value can use general detection method to reappear, to plasmid standards for quantitation using numeral
PCR method determines the frequency of mutation of L858R saltant types, determines 12 repetitions, measurement result such as following table.
The digital pcr measurement result of table 7
The direct uniformity of result is prepared to digital pcr measurement result with gravimetric method to analyze, such as Figure 19.Its orbicular spot
Represent gravimetric method preparation value, triangular representation digital pcr result.Two methods measurement result uniformity is in its range of uncertainty
It is interior fine, but the precision of gravimetric method is better than digital pcr method, shows the use weight Par value EGRF established in the present invention
Detection in Gene Mutation plasmid standards for quantitation, value is accurate, and applicability is good.Checking and performance available for digital pcr or other method
Assess.
Embodiment described above is only that the preferred embodiment of the present invention is described, not to the model of the present invention
Enclose and be defined, on the premise of design spirit of the present invention is not departed from, technical side of the those of ordinary skill in the art to the present invention
The various modifications and improvement that case is made, it all should fall into the protection domain of claims of the present invention determination.
Claims (10)
1. a kind of BRAF and EGFR genetic mutation detection plasmid standards for quantitation preparation method, comprise the following steps：
(1) plasmid containing BRAF and EGFR genetic mutation site is built respectively, is expanded using plasmid as template using regular-PCR
To the genetic fragment containing targeted mutagenesis site, the saltant type and purity for detecting PCR primer are sequenced by Sanger；
(2) wild type human genomic DNA is extracted, carries out fragmentation；The PCR primer of above-mentioned mutation is wild with fragmentation respectively
Type human gene group DNA mixes according to a certain percentage；
(3) add in yeast total serum IgE to the wild type human genomic DNA mixed solution of PCR primer and fragmentation.
2. BRAF according to claim 1 and EGFR genetic mutation detection plasmid standards for quantitation preparation method, its feature exist
In：In the step (2), the wild type human genomic DNA mixed proportion of PCR primer and fragmentation is X:Y, wherein X are 0.01
~5, Y are 5~9.99, and X+Y=10.
3. BRAF according to claim 1 and EGFR genetic mutation detection plasmid standards for quantitation preparation method, its feature exist
In：The constructed plasmid containing BRAF and EGFR genetic mutation site is to be dashed forward respectively containing BRAF gene in the step (1)
Modification V600E, EGFR genetic mutation type T790M, L858R and E746_A750del 4 plasmids.
4. BRAF according to claim 1 or 2 and EGFR genetic mutation detection plasmid standards for quantitation preparation method, its feature
It is：Described segment turns to ultrasonic tear reason；Condition is：Sonicator processing, intensity are focused on using sound wave：5, when
Between：6min, applied sample amount are 100 μ L, DNA concentration：10-50ng/ μ L, working cycles：10%, explosion/circulation：200, temperature：4
℃。
5. BRAF according to claim 1 and EGFR genetic mutation detection plasmid standards for quantitation preparation method, its feature exist
In：In the step (2), the wild type human genomic DNA of PCR primer and fragmentation mixes according to a certain percentage, is to use day
Divide equally after also known as measuring and mixed.
6. BRAF according to claim 1 and EGFR genetic mutation detection plasmid standards for quantitation preparation method, its feature exist
In：Step (3) the yeast total rna concentration is 300~400ng/ μ L.
7. BRAF according to claim 4 and EGFR genetic mutation detection plasmid standards for quantitation preparation method, its feature exist
In：The wild type human genomic DNA length of fragmentation is that scope is distributed as 50bp~320bp using 165bp as major length.
8. the preparation method of the BRAF and EGFR genetic mutation detection plasmid standards for quantitation according to claim any one of 1-5,
It is characterized in that：BRAF and EGFR targeted mutagenesis gene copy numbers density determination method is numeral in PCR primer and wild type DNA
PCR method.
9. BRAF and EGFR genetic mutation the detection plasmid standards for quantitation that any one of claim 6-8 preparation method obtains.
10. the valued methods of the BRAF and EGFR genetic mutation detection plasmid standards for quantitation described in claim 9, it is characterised in that：
Using weight Par value, i.e., the copy Particle density determined according to the quality of weighing and digital pcr, the plasmid standards for quantitation of preparation is entered
Row definite value.
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