CN107663532A - KRAS gene mutation detection plasmid standards for quantitation and preparation method thereof and valued methods - Google Patents
KRAS gene 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 KRAS gene mutation detection plasmid standards for quantitation and preparation method thereof and valued methods, the cell line dna of 7 common mutations types of KRAS genes will be contained as template, enter performing PCR amplification, 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, the plasmid standards for quantitation of 7 kinds of mutation of KRAS genes can be obtained.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 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.This plasmid standards for quantitation can be used for method validation and the quality control of KRAS gene mutation detection method.
Description
Technical field
The present invention relates to field of gene detection, more particularly to a kind of preparation method of KRAS gene mutation examination criteria product
And valued methods.
Background technology
KRAS (K-ras, p21) mrna length is about 35kb, positioned at No. 12 chromosomes, encodes 21kd ras albumen.Normally
KRAS genes participate in Intracellular signals transmission, when KRAS genes are undergone mutation, it is impossible to produce normal ras albumen, can lead
Cause Intracellular signals to transmit disorderly, so as to cause cell continued propagation can not self apoptosis and trigger canceration.In tumor patient
The most common mutation detected is the mutation of 12, No. 13 codons positioned at KRAS genes.It there are about in Pancreas cancer patients
65%~100% patient is the mutation that these sites are carried in KRAS genes, and these mutation are carried in colorectal cancer
Patient accounts for 30%~50%.KRAS gene mutation typically occurs in the early stage that cancer of pancreas and colorectal cancer tumour are formed.Cause
This, KRAS gene mutation helps to make cancer of pancreas and colorectal cancer patients and examined in early days in detection body fluid, pancreatic juice or faeces DNA
It is disconnected.In addition, research shows anti-epidermal growth factor receptor targeted drug (cetuximab and panitumumab) to containing KRAS
The colorectal cancer patients of gene mutation are without therapeutic effect.Therefore, KRAS genes are also used as an anti-epidermal growth factor of screening
The sub- effective mark of receptor target drug therapy.
At present, detecting the method for KRAS gene mutation mainly has Sanger PCR sequencing PCRs and high pass measurement based on sequencing technologies
Sequence method (Next Generation Sequencing, NGS), and the ARMS-PCR (amplification- of PCR-based 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.If generation sequence measurement (Sanger sequencings) is applied to measure allelic mutation when institute energy
The sensitivity 20% or so of measure；And the sensitivity of high-flux sequence method is then in 2-6% or so.The analysis of ARMS-PCR methods
Sensitivity is 1%, and the sensitivity for being mutated enrichment PCR and COLD-PCR is higher, can reach 0.1%.Due to distinct methods
Sensitivity for analysis is different, can cause the inconsistent of testing result and not comparable.Therefore, it is badly in need of the KRAS with accurate mutant proportion
Plasmid standards for quantitation, to examine the comparativity between the sensitivity and specificity of various analysis methods, and various methods.
The country there is no the plasmid standards for quantitation for people's KRAS genetic tests at present.
The content of the invention
, should it is an object of the invention to provide a kind of KRAS gene mutation plasmid standards for quantitation and preparation method thereof and valued methods
The KRAS gene mutation plasmid standards for quantitation degree of accuracy that method obtains is high, with respect to expanded uncertainty 3%.
The present invention establishes the preparation method of KRAS gene mutation plasmid standards for quantitation first, has reached foregoing invention purpose.
The technical scheme is that：
(1) cell line of the extraction containing KRAS gene mutation type (G12A, G12D, G12R, G12C, G12S, G12V, G13D)
Genomic DNA is expanded to obtain the genetic fragment containing targeted mutagenesis site using regular-PCR, surveyed by Sanger as template
The purity in the mutational site of sequence PCR primer.
(2) wild type human genomic DNA is extracted, carries out fragmentation.By the PCR primer of 7 kinds of saltant types and the open country of fragmentation
Raw type human gene group DNA mixes according to a certain percentage.Purpose using wild type human genomic DNA is to simulate dissociating for normal person
DNA, be largely normal cfDNA in general tumour patient, only sub-fraction for mutation ctDNA.
(3) add in yeast total serum IgE to the wild type human genomic DNA mixed solution of PCR primer and fragmentation.
Its specific method is：
Before mixing according to a certain percentage, first by the PCR primer of 7 kinds of saltant types 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 of 7 kinds of saltant types and ultrasonic tear reason fragmentation according to a certain percentage
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 the prior art, generally use micrococcal nuclease (MNase) carries out fragmentation to genome, but uses this method
Afterwards, the enzyme after fragmentation can not complete deactivation, even if using in general inactivation condition (65 degree, 10min) enzyme is carried out
Inactivation, but enzyme does not have in the case of complete deactivation the DNA content of fragmentation can be caused unstable.In addition, needed pair after digestion processing
The DNA of fragmentation is purified, and this process can also largely lose the DNA of fragmentation, therefore this side is used in practical operation
Method needs substantial amounts of DNA sample.And the present invention using sound wave focus on ultrasonic wave fragmentation genomic DNA, without add enzyme or its
His reagent, without purifying after processing, can directly be applied, operation not only simple saving sample again.
In some embodiments, the wild type gene group DNA of PCR primer and fragmentation is diluted to respectively before combination
16000±480copies/μL。
The frequency of mutation value calculation formula in each mutational site is as follows in the plasmid standards for quantitation of the present invention：
Wherein miFor the mass value of the PCR primer containing a certain mutational site, ciCopied for the PCR primer in a certain mutational site
Shellfish Particle density, mWTFor wild type gene group DNA mass value, cWTFor wild type gene group DNA copy Particle density.
In a further preferred embodiment, by containing G12A, G12D, G12R, G12C, G12S, G12V, G13D this 7
The PCR primer of individual mutation and the genomic DNA of fragmentation are X1 according to mixed proportion：X2：X3：X4：X5：X6：X7：Y；Wherein
X1, X2, X3, X4, X5, X6, X7 are that 0.1~50, Y is 50~99.3, and X1+X2+X3+X4+X5+X6+X7+Y=100 ratio
(mass ratio) mixes.
Yeast total rna concentration is 300~400ng/ μ L in a further preferred embodiment.
The KRAS gene mutation detection plasmid standards for quantitation of the present invention carries out definite value using gravimetric method, without external standard, quantitative knot
Fruit can trace 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 people KRAS of the present invention
Gene mutation standard items can be as quantitative standard.The people KRAS standard items of the present invention include 7 kinds of common KRAS positions
Point, available for the assessment and quality control of KRAS gene mutation detection method reliability and dosing accuracy, improve efficiency simultaneously
Reduce 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 KRAS site of pcr amplification product is confirmed simultaneously using Sanger sequence measurements,
As a result the PCR primer of 7 kinds of cell line of discovery is homozygous mutation (being shown in Table 1) by measure.To PCR primer and wild type gene group
DNA purity is determined, and the results are shown in Table 2-9.
The result of the table 1 using Sanger PCR sequencing PCRs to PCR primer saltant type
| Cell line | Sequence |
| G12A | As shown in Figure 1 |
| G12D | As shown in Figure 2 |
| G12R | As shown in Figure 3 |
| G12C | As shown in Figure 4 |
| G12S | As shown in Figure 5 |
| G12V | As shown in Figure 6 |
| G13D | As shown in Figure 7 |
| Wild type | As shown in Figure 8 |
The purity and concentration of table 2.G12A PCR primers
The purity and concentration of table 3.G12D PCR primers
The purity and concentration of table 4.G12R PCR primers
The purity and concentration of table 5.G12C PCR primers
The purity and concentration of table 6.G12S PCR primers
The purity and concentration of table 7.G12V PCR primers
The purity and concentration of table 8.G13D PCR primers
The wild type gene group DNA of table 9. purity and concentration
3. demonstrate gravimetric method preparation and the uniformity of digital pcr method measurement result
G12A PCR primer is mixed such as following table with the wild type gene group DNA of fragmentation according to a certain amount of.Using
Digital pcr method determines six different mutation gradients.By gravimetric method configuration S1 to S6 mutation abundance respectively
19.84%th, 9.87%, 4.94%, 0.98%, 0.19% and 0.10%, the mutation that S1 to S6 is obtained after ddPCR is determined is rich
Degree is respectively 20.81%, 10.57%, 5.32%, 1.05%, 0.21% and 0.10%.The sample of 6 different gradients is by surveying
After fixed, the uniformity between ddPCR and gravimetric method proportioning result is 0.951, sees Figure 10.Hence it is demonstrated that based on gravimetric method configuration
The value of the G12A difference frequencies of mutation can obtain the reproduction of digital pcr method.
The configuration of the different mutation abundance of table 10.G12A and digital pcr method validation result
G12D PCR primer is mixed such as following table with the wild type gene group DNA of fragmentation according to a certain amount of.Using
DdPCR determines five different mutation gradients.By gravimetric method configuration S1 to S5 mutation abundance respectively 21.29%,
10.75%th, 5.41%, 1.06%, 0.10%, the mutation abundance that S1 to S5 is obtained after digital pcr determines is respectively
20.91%th, 10.40%, 5.19%, 1.07% and 0.11%.The sample of 5 different gradients is after measure, ddPCR and weight
Uniformity between method proportioning result is 1.019, sees Figure 11.Hence it is demonstrated that the G12D difference frequencies of mutation based on gravimetric method configuration
Value can obtain the reproduction of digital pcr method.
The configuration of the different mutation abundance of table 11.G12D and ddPCR method validation results
G12R PCR primer is mixed such as following table with the wild type gene group DNA of fragmentation according to a certain amount of.Using
DdPCR determines six different mutation gradients.By gravimetric method configuration S1 to S6 mutation abundance respectively 22.02%,
11.55%th, 6.03%, 1.18%, 0.27% and 0.14%, S1 to S6 mutation abundance difference is obtained after ddPCR is determined
For 21.53%, 11.09%, 5.63%, 1.11%, 0.30 and 0.14%.The sample of 6 different gradients after measure,
Uniformity between ddPCR and gravimetric method proportioning result is 1.024, sees Figure 12, hence it is demonstrated that the G12R based on gravimetric method configuration
The value of the different frequencies of mutation can obtain the reproduction of digital pcr method.
The configuration of the different mutation abundance of table 12.G12R and ddPCR method validation results
G12C PCR primer is mixed such as following table with the wild type gene group DNA of fragmentation according to a certain amount of.Using
DdPCR determines seven different mutation gradients.By gravimetric method configuration S1 to S6 mutation abundance respectively 49.76%,
20.17%th, 10.01%, 5.19%, 0.97%, 0.11% and 0.06%, S1 to S7 mutation is obtained after ddPCR is determined
Abundance is respectively 52.81%, 20.93%, 10.02%, 5.046%, 1.01%, 0.12% and 0.06%.7 different gradients
For sample after measure, the uniformity between ddPCR and gravimetric method proportioning result is 0.941, sees Figure 13.Hence it is demonstrated that based on weight
The value of the G12C difference frequencies of mutation of amount method configuration can obtain the reproduction of digital pcr method.
The configuration of the different mutation abundance of table 13.G12C and ddPCR method validation results
G12S PCR primer is mixed such as following table with the wild type gene group DNA of fragmentation according to a certain amount of.Using
DdPCR determines six different mutation gradients.By gravimetric method configuration S1 to S6 mutation abundance respectively 49.92%,
19.97%th, 9.93%, 5.02%, 1.11% and 0.09%, S1 to S6 mutation abundance difference is obtained after ddPCR is determined
For 50.39%, 20.42%, 9.48%, 5.16%, 1.19% and 0.10%.The sample of 6 different gradients after measure,
Uniformity between ddPCR and gravimetric method proportioning result is 0.989, sees Figure 14.Hence it is demonstrated that the G12S based on gravimetric method configuration
The value of the different frequencies of mutation can obtain the reproduction of digital pcr method.
The configuration of the different mutation abundance of table 14.G12S and ddPCR method validation results
G12V PCR primer is mixed such as following table with the wild type gene group DNA of fragmentation according to a certain amount of.Using
DdPCR determines six different mutation gradients.By gravimetric method configuration S1 to S6 mutation abundance respectively 59.96%,
27.27%th, 14.22%, 7.23%, 1.53% and 0.17%, S1 to S6 mutation abundance difference is obtained after ddPCR is determined
For 58.36%, 25.06%, 13.25%, 7.00%, 1.46% and 0.18%.The sample of 6 different gradients after measure,
Uniformity between ddPCR and gravimetric method proportioning result is 1.03, sees Figure 15.Hence it is demonstrated that the G12V based on gravimetric method configuration is not
Value with the frequency of mutation can obtain the reproduction of digital pcr method.
The configuration of the different mutation abundance of table 15.G12V and ddPCR method validation results
G13D PCR primer is mixed such as following table with the wild type gene group DNA of fragmentation according to a certain amount of.By weight
The S1 to S6 of amount method configuration mutation abundance is respectively 59.96%, 27.27%, 14.22%, 7.23%, 1.53% and
0.17%, the mutation abundance that S1 to S6 is obtained after ddPCR is determined is respectively 58.36%, 25.06%, 13.25%,
7.00%th, 1.46% and 0.18%.The sample of 6 different gradients is after measure, between ddPCR and gravimetric method proportioning result
Uniformity is 1.00, sees Figure 16.Hence it is demonstrated that the value of the G13D difference frequencies of mutation based on gravimetric method configuration can be counted
The reproduction of word PCR method.
The configuration of the different mutation abundance of table 16.G13D and ddPCR method validation results
4. RNA is optimized as protectant addition concentration
Research process of the present invention find that the DNA concentration of DNA especially fragmentations is lower, and the time that can stablize preservation gets over
It is short.Therefore it with the addition of protective agent in the present invention for the DNA plasmid standards for quantitation of fragmentation.Object human genome in the present invention
DNA, corn gene group DNA and yeast total serum IgE have been primarily determined that as protective agent, by expanding the analysis influenceed to PCR, finally
Protective agent of the yeast total serum IgE as DNA plasmid standards for quantitation is determined.And selected for protectant concentration, then separately design
200ng/ μ L, 300ng/ μ L, 400ng/ μ L, 500ng/ng/ μ L, 600ng/ μ L concentration, then enter performing PCR amplification, analysis is not
Positive droplet number is expanded with KRAS gene PCRs under protection agent concentration.When as a result finding that RNA concentration is below 400ng/ μ L, sun
Property number of droplets does not have significant changes, and when adding concentration and reaching 500ng/ μ L and the above, positive droplet number reduces, and this shows
There is inhibitory action to PCR.And it is higher according to protectant addition concentration, more preserved beneficial to the stable of target dna, therefore the present invention
It is final to be determined that the protectant addition concentration of addition RNA is 300-400ng/ μ L.
Compared with the existing technology, its innovative point and advantage are the present invention：
1st, the present invention carries out accurate quantification to KRAS gene mutation standard items using gravimetric method first, while using numeral
PCR method is verified to value, it was demonstrated that its applicability.
2nd, the preparation method of KRAS gene mutation plasmid standards for quantitation has been started, have found suitable ultrasonic wave fragmentation gene
Group DNA condition.
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 wild type gene group DNA of the KRAS gene mutation plasmid standards for quantitation, wherein fragmentation prepared using this method
The dissociative DNA in human blood is simulated, and the PCR primer containing targeted mutagenesis represents Circulating tumor DNA, therefore the standard of the present invention
Product preferably simulate the Circulating tumor DNA sample clinically detected.
6th, it is lower than art methods cost.
Explanation and specific embodiment detect plasmid standards for quantitation to KRAS gene mutation of the present invention below in conjunction with the accompanying drawings
And preparation method thereof and valued methods be described further.
Brief description of the drawings
Fig. 1 is the result of the Sanger PCR sequencing PCRs to G12A.
Fig. 2 is the result of the Sanger PCR sequencing PCRs to G12D.
Fig. 3 is the result of the Sanger PCR sequencing PCRs to G12R.
Fig. 4 is the result of the Sanger PCR sequencing PCRs to G12C.
Fig. 5 is the result of the Sanger PCR sequencing PCRs to G12S.
Fig. 6 is the result of the Sanger PCR sequencing PCRs to G12V.
Fig. 7 is the result of the Sanger PCR sequencing PCRs to G13D.
Fig. 8 is the result of the Sanger PCR sequencing PCRs to wild type.
Fig. 9 is the wild type gene group DNA condition optimizing figures after ultrasonication.
Figure 10 is that G12A is mutated gravimetric method preparation and the uniformity of digital pcr method measurement result.
Figure 11 is that G12D is mutated gravimetric method preparation and the uniformity of digital pcr method measurement result.
Figure 12 is that G12R is mutated gravimetric method preparation and the uniformity of digital pcr method measurement result.
Figure 13 is that G12C is mutated gravimetric method preparation and the uniformity of digital pcr method measurement result.
Figure 14 is that G12S is mutated gravimetric method preparation and the uniformity of digital pcr method measurement result.
Figure 15 is that G12V is mutated gravimetric method preparation and the uniformity of digital pcr method measurement result.
Figure 16 is that G13D is mutated gravimetric method preparation and the uniformity of digital pcr method measurement result.
Figure 17 is the wild type gene group DNA result figures after ultrasonication.
Figure 18 is that digital pcr determines KRAS gene copy number results.
Figure 19 is gravimetric method and digital pcr method comparison of coherence.
Embodiment
Embodiment 1
The preparation of 7 kinds of mutation plasmid standards for quantitation of KRAS genes and definite value
First, materials and methods：
1. cell line and culture medium
RPMI-8226, SUN-C2B, NCI-H157, SW1573, A549, SW620, HCT-116, PG LCL8 are first 7 plants thin
Born of the same parents system is purchased from ATCC, and required culture medium and condition of culture are carried out with reference to ATCC specifications.8th plant of cell line is that Fudan University is big
Learn and provide, culture medium 1640+10%FBS, 5%CO2, 37 degree of cultures.
2.DNA is extracted and PCR amplifications
Use health to extract the genomic DNA of 8 plants of cell line for the Genomic Purification Kit of ShiJi Co., Ltd, contain mesh with 7 kinds
The cell line genomic DNA of mark mutation enters performing PCR amplification for template, obtains the PCR primer containing 7 targeted mutagenesis.PCR is expanded
The μ L of system 20, comprising concentration is 5uM 2 μ L, DNA masterplate of upstream and downstream primer 2 μ L, 2 × PCR premixed liquids 10 μ L, the μ of sterilized water 6
L.Amplification condition is：95 degree, 10min, 40 circulations include 95 degree of 15s, 55 degree, 30s, 72 degree 1min.
3.PCR products and wild type gene group purity test
Using NanoDrop2000 micro ultraviolet specrophotometers measure PCR primer and wild type gene group 260nm,
Light absorption value at 280nm and 230nm, Blank is carried out by the use of TE as blank, then takes 1 μ L samples to be measured.
4. 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.
5. digital pcr method determines KRAS gene copy Particle densities
The copy Particle density of KRAS genes in 7 kinds of PCR primers and wild type gene group DNA is determined using digital pcr method,
Amplification system such as table 17.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 15s, 60 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 17.KRAS gene digital pcr amplification systems
6. gravimetric method configures the plasmid standards for quantitation of 7 kinds of different saltant types
Two mutation frequencies are prepared according to the mass value of table 18 using high-precision 6 assay balances (XP56) by calibration
The plasmid standards for quantitation of rate.
Table 18. is with tabulation
7. digital pcr method validation gravimetric method preparation value
The horizontal plasmid standards for quantitation of the different mutation that gravimetric method prepares is diluted to 5000cp/ μ L or so, according to numeral
The frequency of mutation of each saltant type in the operating procedure measure plasmid standards for quantitation of PCR method,
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 17.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 of 7 kinds of different saltant types the results are shown in Table 1 and Fig. 1-8.As seen from the results in Table 1, often
It is homozygous mutation that saltant type is corresponded in individual PCR primer, and wild type DNA does not contain any mutation.This matches somebody with somebody to plasmid standards for quantitation
Make extremely important.In addition DNA Purities the results are shown in Table 2-8.According to 7 PCR primers of measurement result and wild type gene group
OD260/OD280 is between 1.7~1.9, and OD260/OD230 is more than 2.0, therefore genotype and DNA purity are satisfied by requiring.
3. digital pcr determines KRAS gene copy Particle densities
According to digital pcr measurement result (see Figure 17), it is calculated in every kind of PCR primer and wild type gene group DNA
The copy number of KRAS genes such as table 19.From data in table, the copy Particle density for every kind of saltant type that 7 kinds of PCR primers contain is non-
It is often close..
The copy Particle density of KRAS genes in table 19.PCR products and genomic DNA
4. weight Par value
The copy Particle density determined according to the quality of weighing and digital pcr, definite value is carried out to the plasmid standards for quantitation of preparation, it is fixed
Value the results are shown in Table 20.In 5% horizontal plasmid standards for quantitation, the every kind of mutation of G12A, G12D, G12R, G12C, G12S, G12V, G13D
The frequency of mutation corresponding to type is about 5.02%, 5.02%, 5.02%, 5.05%, 5.00%, 5.00%, 4.99%.1% is horizontal
Plasmid standards for quantitation in, the frequency of mutation corresponding to the every kind of saltant type of G12A, G12D, G12R, G12C, G12S, G12V, G13D is about
1.03%th, 1.02%, 1.01%, 1.01%, 1.00%, 1.00%, 1.01%.
The gravimetric method of table 20. prepares frequency of mutation value
5.dPCR determines the frequency of mutation
In order to verify whether gravimetric method preparation value can use general detection method to reappear, the quantitative criterion horizontal to 2
Product determine the frequency of mutation of every kind of saltant type using digital pcr method, and computational methods are that each saltant type carries out 3 repetitions, are surveyed
Determine result such as following table.
The digital pcr measurement result of table 21.
The direct uniformity of result is prepared to digital pcr measurement result with gravimetric method to analyze, such as Figure 18.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 people established in the present invention
KRAS gene mutation standard items, value is accurate, and applicability is good.Checking and Performance Evaluation available for digital pcr or other method.
Embodiment 2
KRAS gene mutation plasmid standards for quantitation applicability is verified
First, materials and methods：
1. material and reagent
7 kinds of mutation detection kits of people KRAS genes of three kinds of different manufacturers on selection domestic market, random number A,
B、C.The plasmid standards for quantitation (numbering K) and 5% plasmid standards for quantitation (numbering P) that it is 1% that the 7 kinds of mutation of people KRAS genes are horizontal are cloudy
Property reference substance (NC1~NC11), plasmid standards for quantitation and reference substance are prepared by China National Measuring Science Research Inst..
2. instrument and equipment
Quantitative real time PCR Instrument (Roche LC480, Roche LC480 II, AB7500), centrifuge.
3. verify index
The following index of kit is verified using real time fluorescence quantifying PCR method：
1) degree of accuracy：10 part 5% of plasmid standards for quantitation is measured, the frequency of mutation of 5% plasmid standards for quantitation is higher than
The test limit of kit.
2) it is specific：10 parts of negative samples are measured, measurement result should be negative.
3) detection limit：20 part 1% of plasmid standards for quantitation is measured, the frequency of mutation of 1% plasmid standards for quantitation and examination
The detection limit that agent box provides is consistent, and measurement result is positive sample size Ying≤19.
4) it is repeated：By same operating personnel, detection 10 times is repeated to certified reference material using same reagent box, to survey
The relative standard deviation (RSD, %) for measuring result represents.Relative standard deviation is calculated according to formula (1).Relative standard deviation should≤
5%.
2nd, interpretation of result
1. the degree of accuracy
Three kinds of kits use fluorescent quantitative PCR technique, hydrolyze release fluorescence by specific probe, monitoring in real time
The progress of PCR reactions.Qualitative analysis is carried out with the Ct values of each site primer, judges gene mutation situation in sample.But three kinds of examinations
The interpretation standard to mutated-genotype of agent box is different, and B producers are directly judged that A producers and C producers are except Ct values with Ct values
Meet outside requirement, also pay close attention to △ Ct values, that is, be mutated the difference of Ct values (CtM) and external control Ct values (CtW).For B producers,
Ct value≤35, judge that mutation result is the positive, Ct values>38, judge that mutation result is feminine gender, 35<Ct value≤38, if repeating to test
Afterwards, Ct values still within this range, are determined as the doubtful positive.But because sample size limits, it is impossible to repeat to test, therefore be Ct with 37
The critical value of value, Ct values<37 are judged as the positive, and Ct value >=37 are judged as feminine gender.For A producers, G12C, G12R, G12D,
Five sites of G12A, G12S, need to meet Ct values simultaneously<38 and △ Ct<8, ability interpretation is the positive；And two positions of G13D and G12V
Point, Ct values need to be met simultaneously<38 and △ Ct<8, it can just be defined as the positive, remaining situation is feminine gender.For C producers, if △
Ct<8, and meet external control Ct values simultaneously<30, you can it is saltant type to determine sample.
To 7 saltant types of three kinds of kits, 10 part of 5% plasmid standards for quantitation measurement result is the positive, is judged all just
Really, the degree of accuracy 100%.Testing result is shown in Table 22.
22. 3 kinds of kit degree of accuracy testing results of table
| Kit | 12C | 12S | 12R | 12V | 12D | 12A | 13D |
| A | 100.00% | 100.00% | 100.00% | 100.00% | 100.00% | 100.00% | 100.00% |
| B | 100.00% | 100.00% | 100.00% | 100.00% | 100.00% | 100.00% | 100.00% |
| C | 100.00% | 100.00% | 100.00% | 100.00% | 100.00% | 100.00% | 100.00% |
2. detection limit
The kit of three producers claims that detection is limited to 1% mutation level, and actual verification result shows some sites
Detection limit it is not consistent with detection limit that producer declares, the results are shown in Table 23.
For A producers kit, 100% only has been reached to the positive rate in G12A sites, has shown the inspection in G12A sites
Rising limit is consistent with the detection limit that producer declares, and simultaneously the detection limit in remaining 6 site is not 1%.
For B producers kit, this 4 sites of G12C, G12S, G12R, G12A, to 20 times of 1% plasmid standards for quantitation
The result of detection is：Positive rate 100%, show that the detection limit in this 4 sites meets the value that producer declares.But to G12V,
G12D, G13D positive rate are respectively 35%, 60% and 90%, and the detection limit of this 3 points is not 1%, does not meet factory
The value that family declares.
For C producers kit, to G12C, G12S and G12R site, the result of 20 detections of 1% plasmid standards for quantitation
For：Positive rate 100%, the positive rate to G13D sites is 95%, shows G12C, G12S, G12R, G13D this four
The detection in site is limited to 1%, meets the value that producer declares.It is but all to 20 testing results in G12V, G12D, G12A site
False negative, shows the detection limit in these three sites and value that producer declares is inconsistent.
23. 3 kinds of kit detection limit testing results of table
| Kit | 12C | 12S | 12R | 12V | 12D | 12A | 13D |
| A producers | 95.00% | 100.00% | 100.00% | 80.00% | 95.00% | 100.00% | 100.00% |
| B producers | 100.00% | 100.00% | 100.00% | 35.00% | 60.00% | 100.00% | 90.00% |
| C producers | 100.00% | 100.00% | 100.00% | 0.00% | 0.00% | 0.00% | 95.00% |
3. specificity
It is different to the specific testing result of same reagent box different loci, it the results are shown in Table 24.For A producers reagent
Box, 10 parts of negative samples are detected, the testing result in 7 sites is feminine gender.For B producers kit, to 10 parts
Negative sample is detected, and two sites of G12V and G12A judge the false positive for occurring 10%, G12D, G12C, G12S, G12R,
Five sites of G13D are 100% negative.For C producers kit, the testing result in 7 sites is 100% negative.
24. 3 kinds of kit specific detection results of table
4. repeatability
Kit reproducibility is carried out using the horizontal plasmid standards for quantitation of 5% mutation, as a result found for any one
Kit, for any site, the repeatability that three laboratories measure is respectively less than 2%, show high level mutation repeatability compared with
It is good.Reproducibility is carried out using the horizontal plasmid standards for quantitation of 1% mutation, as a result found for any one kit, for
Any site, the repeatability that three laboratories measure are respectively less than 3%.
25. 3 kinds of kit repeatability testing results (%) of table
By experimental verification, the kits of three producers is satisfied by the finger that producer declared in the degree of accuracy and repeatability index
Mark, and for specific index, the only kit of Liang Ge producers meet the requirements；For detection limit, the reagent of three producers
Box can not all meet 7 sites while meet the requirements, and this shows kit producer it is determined that used quality control during detection limit
Typical magnitude processed is not accurate enough.Hence it is demonstrated that the plasmid standards for quantitation obtained using this patent, can meet KRAS genetic tests
Method validation and the requirement of quality control.
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 preparation method of KRAS gene mutation detection plasmid standards for quantitation, comprises the following steps：
(1) the cell line genomic DNA that extraction contains KRAS gene mutation type is expanded using regular-PCR and contained as template
There is the genetic fragment in targeted mutagenesis site, the purity in the mutational site of PCR primer is sequenced by Sanger；The KRAS genes are dashed forward
Modification includes G12A, G12D, G12R, G12C, G12S, G12V, G13D；
(2) wild type human genomic DNA is extracted, carries out fragmentation；By the PCR primer of 7 kinds of saltant types and the wild type of fragmentation
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. the preparation method of KRAS gene mutation detection plasmid standards for quantitation according to claim 1, it is characterised in that：It is described
In step (2), the PCR primer of 7 kinds of saltant types and the wild type human genomic DNA mixed proportion of fragmentation are X1：X2：X3：X4：
X5：X6：X7：Y；Wherein X1, X2, X3, X4, X5, X6, X7 are that 0.1~50, Y is 50~99.3, and X1+X2+X3+X4+X5+X6+
X7+Y=100.
3. the preparation method of KRAS gene mutation detection plasmid standards for quantitation according to claim 1, it is characterised in that：It is described
In step (1), selected cell line for can stablize passage human tumor cell line RPMI-8226, SUN-C2B, NCI-H157,
SW1573、A549、SW620、HCT-116。
4. the preparation method of KRAS gene mutation detection plasmid standards for quantitation according to claim 1 or 2, it is characterised in that：
Described fragmentation refers to make its fragmentation using ultrasonication；Its treatment conditions is to focus on sonicator using sound wave
Processing, intensity：5, the time：6min, applied sample amount are 100 μ L, DNA concentration：10-50ng/ μ L, working cycles：10%, explosion/follow
Ring：200, temperature：4℃.
5. the preparation method of KRAS gene mutation detection plasmid standards for quantitation according to claim 1, it is characterised in that：Step
(2) in, the wild type human genomic DNA of PCR primer and fragmentation mixes according to a certain percentage, is after being weighed respectively using balance
Mixed.
6. the preparation method of KRAS gene mutation detection plasmid standards for quantitation according to claim 1, it is characterised in that：Step
(3) yeast total rna concentration is 300~400ng/ μ L.
7. the preparation method of KRAS gene mutation detection plasmid standards for quantitation according to claim 1, it is characterised in that：Segment
The wild type human genomic DNA length of change is that scope is distributed as 50bp~320bp using 165bp as major length.
8. the preparation method of the KRAS gene mutation detection plasmid standards for quantitation according to any one of Claims 1 to 5, its feature
It is：KRAS targeted mutagenesis gene copy numbers density determination method is digital pcr method in PCR primer and wild type DNA.
9. the KRAS gene mutation detection plasmid standards for quantitation that the preparation method described in any one of claim 6~8 obtains.
10. the valued methods of the KRAS gene mutation detection plasmid standards for quantitation described in claim 9, it is characterised in that：Using weight
Par value is measured, i.e., the copy Particle density determined according to the quality of weighing and digital pcr, the plasmid standards for quantitation of preparation is determined
Value.
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