CN106636404A - Quality control method for detecting human EGFR (Epidermal Growth Factor Receptor) gene variation based on high-throughput sequencing and kit - Google Patents
Quality control method for detecting human EGFR (Epidermal Growth Factor Receptor) gene variation based on high-throughput sequencing and kit Download PDFInfo
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Abstract
The invention discloses a quality control method for detecting human EGFR (Epidermal Growth Factor Receptor) gene variation based on high-throughput sequencing and a kit, and applications thereof. The quality control method comprises the following steps: extracting a plurality of genome DNAs of an EGFR gene variation-positive human tumor cell line; measuring variation positive sites as positive control sites through a Sanger sequencing method; fragmenting the genome DNAs and mixing according to a certain proportion to obtain a quality control product which can be applied high-throughput sequencing detection of human EGFR gene variation. The kit comprises the human EGFR gene variation detection quality control product.
Description
Technical field
The present invention relates to field of gene detection, and in particular to one kind detects people's Circulating tumor DNA EGFR bases based on high flux
Because of the quality control method and kit of variation.
Background technology
Free small pieces segment DNA (cell-free DNA, cfDNA) is there is in blood, and they are thin from death
Born of the same parents.Generally dead cell can be disposed of, therefore the content of cfDNA is low-down, the 1ml blood plasma of a usual Healthy People
In cfDNA containing 25ng.And the content of the cfDNA of cancer patient is higher by normally several times, a portion is ctDNA
(circulating tumor DNA).The relative amount of ctDNA and the load of tumour and the reaction to treating be it is related, can
Drive gene, guiding clinical treatment, monitoring clinical therapeutic efficacy and cancer return, disclose treatment resistance and detection for identification
Progression of disease.Even high traditional means of the sensitivity of ctDNA methods in terms of some.For example, detect with traditional iconography
Compare, follow the trail of the Tumour DNA in blood after early-stage breast cancer operation in patients, 7.9 months can be shifted to an earlier date and find breast cancer relapse.In lung
The EGFR mutation of cfDNA are detected in cancer also has important diagnostic value to lung cancer.CtDNA just can be detected in cancer early stage
Arrive.Because cfDNA is easily collected, show highly consistent with the variation in tissue in lung cancer, therefore the liquid biopsy of ctDNA
Increasingly receive publicity.
CtDNA contents in blood, cancer early stage account for cfDNA ratio be it is very low, typically smaller than 0.1%.CtDNA is examined
Survey method is a lot, such as with digitlization PCR (Digital PCR), or sequencing (Next Generation of future generation
Sequencing, NGS) etc..The deep sequencing (CAPP-seq) for optimizing ctDNA by NGS reaches the susceptibility of abrupt climatic change
0.02%, specificity is 100%.The method combines iDES (the integrated Digital Error for reducing ambient noise
Suppression) after analysis method, the threshold value for making detection frequency is further lowered into 0.004%.So as to substantially increase analysis
The recall rate of CtDNA.
Lung cancer (lung cancer) is most universal and most fatal malignant tumour in global range, and survival rate is only within 5 years
17% or so, the death of 1,600,000 people is caused every year.Chinese smoking and environmental pollution be two topmost lung cancer cause a disease because
Element.There are 600,000 patients with lung cancer dead in China within 2015 according to statistics, increase 730,000 newly.And have growth trend, to possibility in 2025
Cause 1,000,000 people dead.Lung cancer is divided into ED-SCLC (small-cell lung according to histopathologic classification
Carcinomas, SCLC) and non-small cell lung cancer (no-small-cell lung carcinomas, NSCLC), non-small cell
Lung cancer is divided into adenocarcinoma of lung (adenocarcinomas), squamous cell carcinoma (squamous cell carcinomas) and big thin again
Born of the same parents' lung cancer (large-cell carcinomas).Ratio maximum is adenocarcinoma of lung, accounts for 40%, secondly thin to account for 30% squamous
Born of the same parents' cancer, maxicell lung cancer and ED-SCLC respectively account for 15%.
Systemic chemotherapy is always the primary treatment regimen of lung cancer, lacks specificity, large side effects.With in NSCLC
The discovery of some important gene variations, is developed and has been used for clinical treatment for the targeted drug of these genetic mutations.
If Gefitinib is the NSCLC targeted therapy medicines for EGFR (epidermal growth factor receptor) variations
Thing.EGFR plays an important role in cell signalling, once being activated, can cause LCK in tumour cell
Activation and the phosphorylation of itself, so that hyperplasia, transfer, Angiogenesiss and apoptotic suppression.EGFR exon 19
Disappearance and L858R mutation activation EGFR, make the inhibitor medicaments to EGFR more sensitive.Contain in about 50%NSCLC in Asian
Have an EGFR, and 90% be above-mentioned activated form mutation.Therefore the identification of EGFR mutation has very important to treating NSCLC
Effect.Lung cancer and the high-risk people of other associated malignancies can be screened out by the detection to people's Circulating tumor DNA EGFR gene
Group, the early diagnosis beneficial to such disease is treated.
High throughput sequencing technologies (High throughput sequencing) are also known as two generation sequencing technologies (Next
Generation Sequencing, NGS), once parallel sequencing can be carried out to millions of DNA moleculars to hundreds of thousands, so
Sequencing result is compared with reference sequences afterwards, finds abrupt information present on DNA molecular.High throughput sequencing technologies are one
Kind efficiently, accurately detection method of gene mutation.
At present the country there is no the quality-control product and kit that detection is sequenced for people's Circulating tumor DNA EGFR gene.
The content of the invention
It is an object of the invention to provide a kind of quality control method and examination that Human epidermal growth factor receptor genetic mutation is detected based on high-flux sequence
Agent box.
According to an aspect of the present invention, there is provided Human epidermal growth factor receptor genetic mutation detects the preparation method of quality-control product, including following
Step：
(1) human tumor cell line that EGFR gene variation is positive, can stably pass on is selected, genome is extracted and is simultaneously passed through
Sanger PCR sequencing PCRs determine the potential variant sites in each clone in EGFR gene, the heterozygosis that Jing Sanger PCR sequencing PCRs confirm
With homozygosis variant sites as positive control site；
(2) selected human tumor cell line is cultivated, by from the genomic DNA fragment of each clone and according to one
Certainty ratio mixes, and obtains quality-control product.
In some embodiments, " will mix from the genomic DNA fragmentization of each clone and according to a certain percentage
Close " can be the genomic DNA for first extracting each clone, difference fragmentation, then by each clone gene of fragmentation
Group DNA mixes according to a certain percentage.
In some embodiments, " will mix from the genomic DNA fragmentization of each clone and according to a certain percentage
Close " can be the genomic DNA for first extracting each clone, the genomic DNA of each clone is mixed according to a certain percentage,
Then fragmentation.
In some embodiments, " will mix from the genomic DNA fragmentization of each clone and according to a certain percentage
Close " can, first by the genomic DNA fragment of each clone, then extract the fragmentation genomic DNA of each clone, so
The fragmentation genomic DNA of each clone is mixed according to a certain percentage afterwards.
In some embodiments, before mixing according to a certain percentage, first by the fragmentation of each clone or not
The genomic DNA of fragmentation is diluted to same concentrations.
In some embodiments, the fragmentation is by endonuclease bamhi or by ultrasonically treated fragmentation.Excellent
In the embodiment of choosing, DNA fragmentation is made by MNase (micrococcal nuclease) digestion.It is each latent in the quality-control product of the present invention
The variation frequency determination that variation frequency value in variant sites can be determined according to mixed proportion and Jing Sanger PCR sequencing PCRs,
Computing formula is as follows：
Wherein mixed proportion i refers to mixed proportion of the genomic DNA of selected i-th clone in quality-control product,
Variation frequency i refers to variation frequency of the genomic DNA of selected i-th clone on the site.
In preferred embodiments, it is selected stablize the human tumor cell line that passes on be A549, NCI-H720,
NCI-H1650, NCI-H1975, SW48 and SW1417 clone.
In a further preferred embodiment, by A549, NCI-H720, NCI-H1650, NCI-H1975, SW48 and
The genomic DNA of fragmentation the or non-fragmentation that SW1417 clones are obtained is according to 1:1:1:1:1:1 ratio (mass ratio)
Uniform mixing.
It should be appreciated that according to the present invention, the clone for being used includes but is not limited to A549, NCI-H720, NCI-
H1650, NCI-H1975, SW48 and SW1417 clone, it is possible to use other human tumor cell lines.By A549, NCI-H720,
The genomic DNA of fragmentation the or non-fragmentation that NCI-H1650, NCI-H1975, SW48 and SW1417 clone is obtained also may be used
Uniformly to be mixed according to other ratios.The present invention can pass through to choose different clones, configure different fragmentations
Or the ratio of the genomic DNA of non-fragmentation, and verify different variant sites preparing Human epidermal growth factor receptor genetic mutation detection matter
Control product.When the different clones of selection, the ratio of the genomic DNA for preparing different fragmentation or non-fragmentations, obtained
Quality-control product variant sites and/or Mutation frequency may be different, but will not hinder its as quality-control product should
With.
In some embodiments, step (2) is：Selected human tumor cell line is cultivated, the base of each clone is made
Because of a group DNA fragmentation, the then fragmentation genomic DNA of each clone of extraction purification, then by the fragmentation of each clone
Genomic DNA mixes according to a certain percentage, obtains quality-control product.
In some embodiments, the condition of culture of the human tumor cell line is to include 37 DEG C of constant temperature, 5%CO2, humidity
50% passes on when cultivating to cell density up to culture dish area 80-90%.
In some embodiments, making the genomic DNA fragment of each clone includes making each cell by digestion
The genomic DNA fragment of system.In preferred embodiments, the enzyme is MNase (micrococcal nuclease).
In some embodiments, the genomic DNA fragment for making each clone is included in exponential phase cells receipts
Collection cell, abandons supernatant, resuspended with precooling PBS solution, and supernatant is abandoned in suction, then with the resuspended precipitations of 0.1%Triton X-100, on ice
It is centrifuged after incubation, supernatant is abandoned in suction, is subsequently adding BSA and MNase, adds EDTA to stop enzyme reaction after incubation.
In some embodiments, the genomic DNA fragment for making each clone is included in exponential phase cells receipts
Collection, 4 DEG C of centrifugation 5min of speed of 1500g take precipitation and abandon supernatant, then resuspended using precooling PBS solution, 4 DEG C of 5000rpm speed
Supernatant is abandoned in centrifugation 5min, suction；Then the resuspended precipitations of 0.1%Triton X-100 are used, 10min, 5000rpm speed 4 is incubated on ice
DEG C centrifugation 10min；Then inhale and abandon supernatant, add 1X MNase Buffer, resuspended precipitation；Then inhale and abandon supernatant, addition 1 ×
MNase Buffer, resuspended precipitation；1 × BSA and MNase, 37 DEG C of incubation 5min are subsequently adding, add EDTA to stop enzyme reaction.
In some embodiments, fragmentation or the genomic DNA of each clone of non-fragmentation divide before combination
It is not diluted to 15 ± 1ng/ μ L.
In some embodiments, the genome of each clone of fragmentation or non-fragmentation is diluted with TE solution
DNA。
According to another aspect of the present invention, there is provided the Human epidermal growth factor receptor genetic mutation obtained by said method detects Quality Control
Product.
According to another aspect of the present invention, there is provided Human epidermal growth factor receptor genetic mutation detection kit, wherein comprising the present invention's
Human epidermal growth factor receptor genetic mutation detects quality-control product.
The Human epidermal growth factor receptor genetic mutation detection kit of the present invention can also include the high flux for Human epidermal growth factor receptor genetic mutation
Any other component of sequencing detection.In preferred embodiments, the Human epidermal growth factor receptor genetic mutation detection kit is further
Reaction buffer, DNA ligase, coupled reaction buffer solution, mark containing molecule are repaired comprising enzymatic mixture, end is repaired selected from end
The joint of label, amplified library primer, PCR premixed liquids, tab closure agent, DNA sealers, hybridization buffer, hybridization enhancers, magnetic
Pearl washing lotion, hybridization washing lotion, capture library PCR primer, capture probe, nucleic acid purification magnetic bead, one kind of streptavidin magnetic bead or
Plurality of reagents.The Human epidermal growth factor receptor genetic mutation detection kit of the present invention optionally can also be comprising negative quality-control product.Especially excellent
In the embodiment of choosing, the Human epidermal growth factor receptor genetic mutation detection kit includes above-mentioned whole reagents.
According to another aspect of the invention, there is provided above-mentioned Human epidermal growth factor receptor genetic mutation detection quality-control product or Human epidermal growth factor receptor genetic mutation
Application of the detection kit in the quality control that the high-flux sequence of Human epidermal growth factor receptor genetic mutation is detected, it is particularly swollen in people's circulation
Application in the quality control of the high-flux sequence detection of knurl DNA EGFR gene variation.
In the present invention, detection Human epidermal growth factor receptor genetic mutation can include the EGFR gene variation of detection human genome, can also wrap
Include the EGFR gene variation of detection people's Circulating tumor DNA.
" high-flux sequence " of the present invention is also referred to as the sequencing of two generations, and it is mainly characterized by can simultaneously to input
Sequence carries out large-scale parallel sequencing, obtains the sequencing result of a large amount of short sequences.The general principle of high-flux sequence is will be to be measured
DNA is broken at random small fragment, and the step such as the reparation of Jing ends, jointing sequence, PCR carries out library construction, finally uses
The sequenators such as Illumina, Ion Torrent are sequenced.
The Human epidermal growth factor receptor quality-control product of the present invention can be used as positive quality control product.The Human epidermal growth factor receptor quality-control product of the present invention is comprising different
The variant sites of variation frequency, are conducive to controlling precision of analysis.The Human epidermal growth factor receptor genetic mutation detection quality-control product that the present invention is provided
Effectively the high pass of Human epidermal growth factor receptor genetic mutation (particularly people's Circulating tumor DNA EGFR gene variation) can be measured with kit
The reliability of sequence detection architecture is estimated, and improves efficiency and reduces detecting Quality Control cost.
Description of the drawings
Fig. 1 is the electrophoresis detection figure of quality-control product.
Specific embodiment
Test method used in following embodiments if no special instructions, is conventional method.
Material and reagent used in following embodiments, if no special instructions, can obtain from commercial channels.
Embodiment 1：The preparation of people's Circulating tumor DNA EGFR gene variation detection quality-control product
1.1. six plants of conventional stablizing are obtained from ATCC purchases and passes on human tumor cell line A549, NCI-H720, NCI-
H1650, NCI-H1975, SW48 and SW1417.The genomic DNA of each clone is sequenced with Sanger methods, Jing
The heterozygosis and homozygosis variant sites of Sanger methods confirmation is used as positive control site.Empirical tests are altogether containing 12 positive change dystopys
Point.
1.2. using this six plants of human tumor cell lines of special culture media culture, condition of culture：37 DEG C of constant temperature, 5%CO2 is wet
Degree 50%.Cultivate and passed on when reaching culture dish area 80-90% to cell density, collect in exponential phase cells, the speed of 1500g
4 DEG C of centrifugation 5min of degree take precipitation and abandon supernatant.
1.3. resuspended using precooling PBS solution, supernatant is abandoned in 4 DEG C of centrifugation 5min of 5000rpm speed, suction.Repeat this step one
It is secondary.
1.4. inhale and abandon supernatant, using the resuspended precipitations of 0.1%Triton X-100,10min, 5000rpm speed 4 are incubated on ice
DEG C centrifugation 10min.Supernatant is abandoned in suction, adds 1X MNase Buffer, resuspended precipitation.
1.5. inhale and abandon supernatant, add 1 × MNase Buffer, resuspended precipitation.Add 1 × BSA and MNase, 37 DEG C of incubations
5min.EDTA is added to stop enzyme reaction.Supernatant is taken after 2000g centrifugation 1min to extract using Life Tech MagMax kits,
The six plants of human tumor cell line nucleic acid TE solution for most extracting at last are diluted to 15 ± 1ng/ μ L, and equal proportion mixes, -20 DEG C of guarantors
Deposit.Take a small amount of equal proportion mixture to useGX Touch and supporting DNA High Sensitivity
Reagent Kit (the two is purchased from PerkinElmer, Inc.) carry out electrophoresis detection, and by specification is operated.Testing result
As shown in Figure 1.Electrophoresis result shows：Size concentrates on 143bp after quality-control product fragmentation, is close to cfDNA sizes in human body, reaches
Prepare to quality-control product and require.
Wherein MNase is micrococcal nuclease, and 1X MNase Buffer and MNase are purchased from NEB.BSA is the clear egg of cow's serum
In vain, purchased from NEB.
Embodiment 2：The preparation of people's Circulating tumor DNA EGFR gene mutation test kit
2.1. the multiple capture probes for different target region in people's Circulating tumor DNA EGFR gene are designed and synthesized,
The set of all capture probes can cover Human epidermal growth factor receptor gene whole encoded exon region and extron introne juncture area；
There is biotin labeling on capture probe；The for example following middle SEQ ID NO of the sequence of the capture probe:Shown in 1-195.
2.2. by SEQ ID NO in upper table:Whole capture probes shown in 1-195 are mixed by same ratio, and
Mixture is diluted into working concentration 1.5PM (PM=picomoles/liter), -20 DEG C of preservations.
2.3. the quality-control product obtained in capture probe mixture and embodiment 1 is dispensed respectively.
2.4. specification, external packing, assembling sealing are prepared.
2.5. wherein the consumption of capture probe mixture is 6 μ L/3 reactions, and 12 μ L/6 reactions, 24 μ L/12 react, 48 μ L/
24 reactions；The consumption of quality-control product is 5 μ L/3 reactions, 10 μ L/6 reactions, 20 μ L/12 reactions, 40 μ L/24 reactions.
Embodiment 3：The sequencing detection of people's Circulating tumor DNA EGFR gene variation
It is gene order automatic analyzer CN500 that used instrument is sequenced in the present embodiment.
The preparation method of quality-control product is with embodiment 1.
3.1. people cfDNA is extracted from 15 positive plasma samples, quality-control product is directly used, without the need for extracting.
3.2. library construction
3.2.1. end is repaired
0.2ml PCR reaction tubes, the cfDNA samples or quality-control product after adding 30ng to extract, with Low EDTA TE polishings extremely
50 μ L, concussion is mixed, of short duration centrifugation.It is separately added into 1 μ L ends and repairs enzymatic mixture and 6 μ L ends reparation reaction buffer, shake
Swing and be incubated 5min at 37 DEG C after the of short duration centrifugation of mixing, 30min, 37 DEG C of insulation 5min are subsequently incubated at 65 DEG C, carry out end reparation.
Reaction takes out PCR reaction tubes after terminating, and product is fully transferred in new 1.5mL centrifuge tubes；Add in each centrifuge tube
108 μ L nucleic acid purification magnetic beads, after fully mixing 5min is incubated at room temperature.Centrifuge tube is transferred on magnetic frame, 2min is stored at room temperature
Afterwards, careful suction abandons supernatant.The ethanol of 200 μ L 80% is added, after being stored at room temperature 30s, careful suction abandons supernatant, repeats once, really
Protect no liquid residual.Centrifuge tube is maintained on magnetic frame, drying at room temperature 5min.30 μ L are separately added into each centrifuge tube
Low EDTA TE, 18 μ L buffer solutions, 1 μ L repair enzymes G3,1 μ L repair enzymes G4, after fully mixing, in 20 DEG C 20min are incubated.Take
Go out centrifuge tube, be separately added into 50 μ L PEG NaCl, piping and druming is incubated at room temperature 5min after mixing.Centrifuge tube is transferred on magnetic frame,
After being stored at room temperature 2min, careful suction abandons supernatant.The ethanol of 200 μ L 80% is added, after being stored at room temperature 30s, careful suction abandons supernatant.Weight
Multiple operation is once, it is ensured that no liquid is remained.Centrifuge tube is maintained on magnetic frame, drying at room temperature 5min.
3.2.2. joint connection
5 μ L joints are added separately to in dried centrifuge tube, and carry out mark；It is separately added into 20 μ L Low
EDTA TE, 3 μ L joint buffer solutions, 2 μ L ligases, fully mix and after resuspended magnetic bead, 25 DEG C of constant-temperature incubation 15min.Add
49.5 μ L PEG NaCl, after fully mixing 5min is incubated at room temperature.Centrifuge tube is transferred on magnetic frame and stands 2min, it is careful to inhale
Abandon supernatant.The ethanol of 200 μ L 80% is added, after being stored at room temperature 30s, careful suction abandons supernatant, repeats once, it is ensured that no liquid
Residual.Centrifuge tube is maintained on magnetic frame, drying at room temperature 5min.Be separately added into 30 μ L Low EDTA TE, 16 μ L buffer solutions,
3 μ L ligases, fully mix and after resuspended magnetic bead, 40 DEG C of constant-temperature incubation 10min.82.5 μ L PEG NaCl are added, is fully mixed
After be incubated at room temperature 5min.Centrifuge tube is transferred on magnetic frame and stands 2min, careful suction abandons supernatant.Add the second of 200 μ L 80%
Alcohol, after being stored at room temperature 30s, careful suction abandons supernatant, repeats once, it is ensured that no liquid is remained, and drying at room temperature 5min (can not make
Magnetic bead is over-drying).20 μ L Low EDTA TE are added, 3min is incubated at room temperature.Centrifuge tube is transferred on magnetic frame, is stood
2min, careful supernatant of drawing is into new 1.5mL centrifuge tubes.
3.2.3 amplified library with purifying
It is separately added into 25 μ L PCR amplification enzymatic mixtures, 5 μ L primer mixtures and enters performing PCR amplification.To reaction complete from
In heart pipe, add 1.65 times of PEG NaCl solutions, and piping and druming to be well mixed, be incubated at room temperature.After the completion of incubation, centrifuge tube is put
2-3min is stood on magnetic frame, after solution clarification, solution is abandoned in suction, and period never contacts magnetic bead.Add in centrifuge tube new
The fresh μ L of 80% ethanol 200, after standing 30s, ethanol is abandoned in suction, is sure not to contact magnetic bead, is repeated once.Suction is abandoned after ethanol, will
Magnetic bead is dried to showing in frosted shape, is sure not to make magnetic bead overdrying, many cracks occurs, after magnetic bead dries, adds Low EDTA TE
The μ L of buffer 20, and be incubated at room temperature.After the completion of incubation, it is placed on magnetic frame, stands 2-3min, after solution clarification, draws molten
Liquid, in being transferred to new 1.5mL LoBind centrifuge tubes.
3.3. library capture
3.3.1. hybridize
1-6 library is blended in a sample cell carries out hybrid capture, and library is mixed according to quantity 1.5mL
Become a sample cell in LoBind centrifuge tubes, sample consumption is not less than 125ng, and sample cell total amount is less than 1 μ g.To sample cell
2 μ L tab closures agent of middle addition and 5 μ L DNA sealers, concussion is mixed, and is freeze-dried under vacuum after of short duration centrifugation.Add 8.5 μ L
Hybridization buffer (2 ×), 2.7 μ L hybridization enhancers and 3.8 μ L PCR-grade water, concussion is mixed, after of short duration centrifugation, room
It is molten that temperature stands 5min weights.Liquid of the weight after molten is fully transferred in new 0.2mL PCR reaction tubes, 95 DEG C of insulation 10min.Take
Go out PCR reaction tubes, add after of short duration centrifugation 2 μ L embodiments 2 obtain capture probe mixture, concussion mix 3-5s, it is of short duration from
More than 4h is incubated at 68 DEG C after the heart, hot lid temperature is 78 DEG C.
3.3.2. it is enriched with
Take and add 1 times of volume magnetic bead washing lotion after the washing of streptavidin magnetic bead, be dispensed into by the μ L of every pipe 100 after vibration is resuspended
In different 0.2mL PCR reaction tubes.
PCR reaction tubes in step 3.3.1 are taken out, whole hybrid products are transferred to containing strepto- parent after of short duration centrifugation
In the PCR reaction tubes of biscuit porcelain pearl, fully suspend 65 DEG C of incubation 45min in PCR instrument, and period inhales every 12min and plays mixing
Once.After incubation terminates, often pipe adds the 1 × washing lotion I of 100 μ L, 65 DEG C of preheatings, and whole suspensions are transferred into 1.5mL LoBind
In centrifuge tube, concussion is mixed, and is transferred to after of short duration centrifugation on magnetic frame and is stood 20s, and supernatant is abandoned in suction.
1 × hybridization washing lotion of 200 μ L, 65 DEG C of preheatings, concussion is added to mix, 65 DEG C of incubation 5min, transfer after of short duration centrifugation
20s is stood to magnetic frame, supernatant is abandoned in suction.Repeat this step once.
18 μ L Nuclease-free water are added after fully being eluted with magnetic bead washing lotion again, resuspended rear all transfers are shaken
It is standby into 0.2mL PCR reaction tubes.
3.3.3. amplified library is captured with purifying
25 μ 2 × PCR of L premixed liquids and each 2.5 μ L of capture library PCR primer are separately added into in above-mentioned PCR reaction tubes, are filled
Divide to mix and enter performing PCR amplification by following programs after of short duration centrifugation：95℃3min；10 circulation, each circulation be 98 DEG C of 20s, 60
DEG C 30s, 72 DEG C of 30s；72℃1min；4 DEG C save backup.
After by the of short duration centrifugation of PCR reaction tubes, product is fully transferred in 1.5mL centrifuge tubes.Add 75 μ L nucleic acid purifications
Magnetic bead, concussion is mixed, and is stored at room temperature of short duration centrifugation after 10min, is transferred on magnetic frame and is stood 1min, and supernatant is abandoned in suction.Add 200
The ethanol of μ L 80%, after standing 30s, supernatant is abandoned in suction.Repeated washing once, is uncapped and is dried to dripless residual.Add 21.6 μ L
Nuclease-free water shake resuspended magnetic bead, are stored at room temperature 5min, are transferred on magnetic frame and stand 1min, take on 20 μ L
It is clear to standby in new 1.5mL LoBind centrifuge tubes.Taking the quality-control product DNA in 2 μ L capture libraries or capture carries out quantitatively (using
Qubit fluorescent quantitation instruments).
3.4. sequencing and data analysis
3.4.1. it is sequenced
Upper machine sequencing is carried out according to sequencing instrument and matched reagent operation instruction.Average effective overburden depth >=5000 ×.
3.4.2. data analysis
After obtaining raw sequencing data, compare with reference data storehouse, carry out analysis of variance and deciphering.
Testing result is as follows：
The mutational site of clinical sample and testing result are as shown in table 1.Clinical sample detects coincidence rate：15/15=
100%.All clinical sample Jing digital pcrs method checkings are errorless.
The clinical sample mutational site of table 1 and testing result
The variant sites testing result of quality-control product as shown in table 2, wherein with reference to the frequency of mutation be according to mixed proportion and
It is high-flux sequence laboratory test results that the variation frequency that Jing digital pcrs method is determined calculates the theoretical detected value for obtaining.
The quality-control product variant sites of table 2 and testing result
| No. | Gene | CDNA variation information | Protein variant information | Reference frequency | Testing result |
| 1 | EGFR | c.T2709C | p.T903T | 67.57% | 65.38% |
| 2 | EGFR | c.C474T | p.N158N | 55.70% | 56.96% |
| 3 | EGFR | c.G2361A | p.Q787Q | 57.13% | 51.11% |
| 4 | EGFR | c.G1562A | p.R521K | 30.05% | 29.71% |
| 5 | EGFR | c.T1887A | p.T629T | 55.47% | 58.24% |
| 6 | EGFR | c.C2047T | p.L683L | 8.05% | 8.26% |
| 7 | EGFR | c.2235_2249del | p.745_750del | 9.17% | 17.69% |
| 8 | EGFR | c.C1839T | p.A613A | 16.65% | 15.77% |
| 9 | EGFR | c.C2369T | p.T790M | 11.92% | 12.38 |
| 10 | EGFR | c.G2028A | p.P676P | 4.42% | 4.59% |
| 11 | EGFR | c.T2573G | p.L858R | 11.93% | 13.57% |
| 12 | EGFR | c.G2155A | p.G719S | 5.30% | 5.28% |
From the result of table 2, variant sites detection coincidence rate is 100% in quality-control product.
Embodiment 4：Repeatability experiment
Preparation method according to embodiment 1 prepares quality-control product E-P2.
Three batches of kits are produced according to the preparation method of embodiment 2 using quality-control product E-P2, lot number is respectively 20160314,
20160315,20160316.
Detection method according to embodiment 3 carries out high-flux sequence detection.Same batch kit 20160316, detects E-
P2 tri- times, testing result is shown in Table 3.Different batches kit 20160314,20160315,20160316, E-P2 is each three times for detection,
Testing result is shown in Table 4.
Difference in 3 batches, table
| Lot number | Sample names | Positive variation coincidence rate |
| 20160316 | E-P2 | 100% (12/12) |
| 20160316 | E-P2 | 100% (12/12) |
| 20160316 | E-P2 | 100% (12/12) |
The differences between batches of table 4
| Lot number | Sample names | Positive variation coincidence rate |
| 20160314 | E-P2 | 100% (12/12) |
| 20160315 | E-P2 | 100% (12/12) |
| 20160316 | E-P2 | 100% (12/12) |
Testing result：Kit is repeatable to meet requirement.
Claims (10)
1. Human epidermal growth factor receptor genetic mutation detects the preparation method of quality-control product, comprises the following steps：
(1) human tumor cell line that EGFR gene variation is positive, can stably pass on is selected, genome is extracted and is simultaneously passed through Sanger
PCR sequencing PCR determines the potential variant sites in each clone in EGFR gene, heterozygosis and homozygosis that Jing Sanger PCR sequencing PCRs confirm
Variant sites are used as positive control site；
(2) selected human tumor cell line is cultivated, by from the genomic DNA fragment of each clone and according to certain ratio
Example mixing, obtains quality-control product.
2. preparation method according to claim 1, wherein step (2) " by from the genomic DNA fragment of each clone
And mix according to a certain percentage " it is the genomic DNA for first extracting each clone, difference fragmentation, then by each of fragmentation
Individual clone genomic DNA mixes according to a certain percentage；Or the genomic DNA of each clone is first extracted, each is thin
The genomic DNA of born of the same parents system mixes according to a certain percentage, then fragmentation；Or first by the genomic DNA piece of each clone
Duan Hua, then extract the fragmentation genomic DNA of each clone, then by the fragmentation genomic DNA of each clone according to
Certain proportion mixes.
3. according to the preparation method of claim 1 or 2, wherein it is selected stablize the human tumor cell line that passes on be A549,
NCI-H720, NCI-H1650, NCI-H1975, SW48 and SW1417 clone.
4. preparation method according to claim 3, wherein will be by A549, NCI-H720, NCI-H1650, NCI- in step (2)
The genomic DNA of fragmentation the or non-fragmentation that H1975, SW48 and SW1417 clone is obtained is according to 1:1:1:1:1:1
Mass ratio uniformly mixes.
5. the Human epidermal growth factor receptor genetic mutation detection quality-control product for being obtained by the preparation method of any one of claim 1-4.
6. Human epidermal growth factor receptor genetic mutation detection kit, wherein the quality-control product comprising claim 5.
7. the kit of claim 6, the kit further repairs reaction comprising repairing enzymatic mixture, end selected from end
Buffer solution, DNA ligase, coupled reaction buffer solution, the joint containing molecular label, amplified library primer, PCR premixed liquids, joint
Sealer, DNA sealers, hybridization buffer, hybridization enhancers, magnetic bead washing lotion, hybridization washing lotion, capture library PCR primer, capture
One or more reagent of probe, nucleic acid purification magnetic bead and streptavidin magnetic bead.
8. the kit of claim 6, the kit further repairs enzymatic mixture, end and repairs reaction buffering comprising end
Liquid, DNA ligase, coupled reaction buffer solution, the joint containing molecular label, amplified library primer, PCR premixed liquids, tab closure
Agent, DNA sealers, hybridization buffer, hybridization enhancers, magnetic bead washing lotion, hybridization washing lotion, capture library PCR primer, capture are visited
Pin, nucleic acid purification magnetic bead and streptavidin magnetic bead.
9. the kit of the quality-control product of claim 5 and any one of claim 6-8 is measured in the high pass of Human epidermal growth factor receptor genetic mutation
Application in the quality control of sequence detection.
10. the kit of the quality-control product of claim 5 and any one of claim 6-8 becomes in people's Circulating tumor DNA EGFR gene
Application in the quality control of different high-flux sequence detection.
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