CN106801091A - The kit and reaction system of the deletion mutation of detection human EGFR gene exons 19 - Google Patents
The kit and reaction system of the deletion mutation of detection human EGFR gene exons 19 Download PDFInfo
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Abstract
The invention discloses a kind of kit and reaction system for detecting the deletion mutation of human EGFR gene exons 19, the kit includes：Sense primer, anti-sense primer, detection the deletion mutation of exons 19 fluorescence probe and detection amplification after STb gene fluorescence probe.The kit devises the primer pair of particular sequence and the probe of particular sequence for the deletion mutation of EGFR gene exons 19, by the method for digital PCR platforms in the reaction system of optimization, realize the high-sensitivity detection to the deletion mutation of EGFR gene exons 19, the other detection can simultaneously detect 18 kinds of different types of nicked forms of EGFR gene exons 19, versatility is substantially increased, so that for EGFR targeted therapies provide reference；Detectable samples sources are various in further the application, both can be tumor tissues, or ctDNA, expand the use scope of the kit, reaction system and method.
Description
Technical field
The present invention relates to a kind of kit for detecting gene mutation, reaction system and method, more particularly to a kind of detection people
The kit of the deletion mutation of class EGFR gene exons 19, reaction system and method.
Background technology
Lung cancer all occupies the first place of global cancer related mortality all the time.IARC of the World Health Organization is sent out
The cancer report display of cloth：Global lung cancer new cases predict about 1,610,000, and death about 1,380,000 accounts for malignant tumour new respectively
The 13% and 18% of morbidity example and death, occupies malignant tumour first, wherein non-small cell lung cancer (Non-Small Cell
Lung Cancer, NSCLC) account for more than the 80% of lung cancer.Although the diagnostic method of lung cancer and treatment means are improved constantly, lung cancer
The death rate be not effectively controlled yet, the prognosis of patient is poor, and 5 years survival rates are still<20%.
Human epidermal growth factor acceptor family (epidermal growth factor receptor family, EGFR
Family) belong to tyrosine kinase receptor family, it is a kind of multi-functional glycoprotein, it is distributed widely in each cell membranes in tissue of human body
On, it is the expression product of proto-oncogene, its gene is located on No. seven the short arm of a chromosome (7p12), by 188.307K base group
Into, 28 extrons are had, EGFR has EGFR-TK (tyrosine kinase, TK) area, and it is that transmission extracellular signal is arrived
Intracellular it is important by way of albumen, and encoded by exons 1 8.24.The structure of EGFR family members is similar, all including extracellular fragment, across
Three parts of film area and intracellular section.Extracellular fragment is binding of receptor and ligand portion, and cross-film section is hydrophobic portion, and acceptor is fixed on into cell membrane
On, intracellular region has typical ATP-binding site and EGFR-TK area, and its tyrosine kinase activity is in regulation cell propagation and divides
Vital effect is played in change.After part is with acceptor combination, cause the dimerization of acceptor to act on, form homotype or abnormal shape
Dimer, the acceptor of dimerization crosslinks specific tyrosine residue on the acceptor of phosphorylation, i.e., one and another acceptor
Phosphorylation, activates the TK subprovinces of intracellular region, so as to excite next stage signal transduction.Non-small cell lung cancer EGFR mutation more than 85%
There is the deletion mutation and the L858R point mutation of exon 21 in extron 19.
In recent years, EGFR (EGFR) tyrosine kinase inhibitors (TKIs) Gefitinib, E Luo
It is proved to advanced Non-small cell lung (NSCLC) patient with EGFR activated mutants with notable for Buddhist nun and Afatinib etc.
Curative effect, EGFR-TKI turns into the milestone of Treatment for Non-small Cell Lung.But meanwhile, EGFR-TKI is reacted when nearly all initial
Good patient resistance, progression of disease can occur in 6-12 month, referred to as " acquired resistance ".
The mutation status that many researchs have proven to EGFR determine reaction of the tumour to EGFR-TKI, and research shows EGFR-
TKI medicines are notable for patient's effect of the deletion mutation of EGFR L858R and EGFR exons 19, especially show outside EGFR gene
There is the Patients with Non-small-cell Lung of deletion mutation to such medicaments insensitive, i.e. receptor tyrosine kinase inhibitors medicine pair in son 19
These patients are effective in cure.Therefore EGFR extron exon19 deletion mutations are accurately detected, can be screening targeted therapy patient
Reference is provided；Can be used for the high sensitivity early stage recurrence monitoring of cancer patient, particularly patients with lung cancer simultaneously, and during medication
Resistance mutation is monitored.But the deletion mutation of exons 19 is mainly 9,12,15,18,24 in-frame deletions of nucleotides of generation and dashes forward
Become, its nicked forms is more, to detect that its deletion condition brings certain difficulty.
The most frequently used samples sources of detection EGFR genetic mutation are the pathological tissue or cytologic specimen of tumour at present, but
Tissue sampling is invasive operation, is difficult to carry out sometimes, and these operations are by shadows such as tumor size, position, patient's ordinary circumstances
Ring, satisfied tissue or tissue mass can not be obtained sometimes can not carry out detection in Gene Mutation very little.And with the progress of disease
With the appearance of EGFR-TKIs drug resistances, it is often necessary to carry out molecular Biological Detection again, and tissue sampling is invasive operation,
Can not it is convenient, be repeated.Plasma DNA (ctDNA) was considered as a kind of possible replacement tumor tissues for examining in recent years
Survey the sample that tomour specific is sexually revised.Tumor tissues, tumor cell necrosis and the tumour cell for coming off are released into apoptosis after blood
Put dissociative DNA and enter peripheral blood.CtDNA is used for Tumor mutations detection advantage：Operation is noninvasive；In any process of disease
Can all obtain；Real-time detection and dynamic monitoring can be realized as a kind of tumor marker；Overcome the heterogeneity of tumor tissues.
Therefore, patient's EGFR genetic mutation state is monitored using peripheral blood dissociative DNA, explores patient EGFR-TKI resistance mechanisms and prediction
Prognosis turns into a feasible approach.However, still thering are some technical to choose using ctDNA detections EGFR genetic mutation at present
War.1) ctDNA contents vary with each individual, and most people content is relatively low；2) ctDNA fragments are relatively small, about 180bp：3)
The related DNA proportion different people difference of tumour is larger in ctDNA, and often is difficult to measure because ratio is small；4) in the past
Research display, compared with tumor tissues, ctDNA detection EGFR mutation sensitivity is only 43-66%.Therefore, it is highly sensitive
The development of ctDNA EGFR genetic mutation detection techniques is particularly significant.
In sum, this area in the urgent need to one kind can be based on nonneoplastic tissue, high sensitivity, high accuracy and Gao Ke
The method that the deletion mutation of EGFR exons 1s 9 in patient is detected by property.
The information for being disclosed in the background section is merely intended to increase the understanding to general background of the invention, without answering
In being considered as recognizing or imply in any form that the information structure has been the prior art well known to persons skilled in the art.
The content of the invention
It is an object of the invention to provide a kind of kit for detecting human EGFR gene mutations, reaction system and method,
The kit devises the primer pair of particular sequence and the probe of particular sequence for the deletion mutation of EGFR gene exons 19,
By the method for Thermo QuantStudio 3D digital PCR platforms in the reaction system of optimization, realize to EGFR bases
Because of the high-sensitivity detection of the deletion mutation of exons 19；And detecting the yin and yang attribute of the deletion mutation of EGFR gene exons 19
The ratio of the deletion mutation of EGFR gene exons 19 in sample can also be obtained simultaneously；The other detection can simultaneously detect 18 kinds
The different types of nicked forms of EGFR gene exons 19, substantially increase versatility, so that for EGFR targeted therapies provide ginseng
Examine；Detectable samples sources are various in further the application, both can be tumor tissues, or ctDNA, expand
The use scope of the kit, reaction system and method.
Concrete technical scheme of the invention is as follows：
A kind of kit for detecting the deletion mutation of human EGFR gene exons 19, including：With such as SEQ ID NO:1 institute
Show the sense primer of sequence, with such as SEQ ID NO:The anti-sense primer of sequence shown in 2, with such as SEQ ID NO:Sequence shown in 3
Row detection the deletion mutation of exons 19 fluorescence probe and with such as SEQ ID NO:It is total after the detection amplification of sequence shown in 4
The fluorescence probe of DNA.Sense primer SEQ ID NO:1 sequence is GAAAGTTAAAATTCCCGTCGCT, anti-sense primer SEQ
ID NO:2 sequence is AGCAGAAACTCACATCGAGG.Detect the fluorescence probe sequence SEQ ID of the deletion mutation of exons 19
NO:3 is TCCTTGTTGGCTTTC, the fluorescence probe sequence SEQ ID NO of STb gene after detection amplification:4 are
AGGAATTAAGAGAAGCAAC.Detect that the end of fluorescence probe 5 ' of the deletion mutation of exons 19 is connected with fluorescent reporter group, preferably
It is VIC, detects that the end of fluorescence probe 3 ' of the deletion mutation of exons 19 is connected with quenching group, preferably MGB；It is total after detection amplification
The end of fluorescence probe 5 ' of DNA is connected with fluorescent reporter group, and the fluorescent reporter group is different from the detection missing of exons 19 and dashes forward
Fluorescent reporter group in the fluorescence probe of change, preferably FAM, the end of fluorescence probe 3 ' of STb gene is connected with and quenches after detection amplification
Go out the group of connecing, preferably MGB.
Mentioned reagent box is in another implementation, and the sense primer, anti-sense primer, the detection missing of exons 19 are prominent
The fluorescence probe of STb gene exists in the form of primed probe mixed liquor after the fluorescence probe of change and detection amplification, i.e., it is described on
The fluorescence probe for swimming STb gene after primer, anti-sense primer, the fluorescence probe of the detection deletion mutation of exons 19 and detection are expanded is molten
In same TE buffer solutions, concentration of the sense primer in primed probe mixed liquor is 12 μM to solution, and the anti-sense primer exists
Concentration in primed probe mixed liquor is 12 μM, and the fluorescence probe of the detection deletion mutation of exons 19 mixes in primed probe
Concentration in liquid is 3 μM, and concentration of the fluorescence probe of STb gene in primed probe mixed liquor is 3 μM after the detection amplification.
Mentioned reagent box in another implementation, also including digital pcr reaction premixed liquid, preferably
QuantStudioTM3D Digital PCR Master Mix v2。
In another implementation, also including positive quality control product, the positive quality control product is by following for mentioned reagent box
Mode is prepared：The deletion mutation cell line genomic DNA of EGFR exons 1s 9 is mixed with wild-type cell system genomic DNA
The DNA ratios of the deletion mutation of exons 1 containing EGFR gene 9 is accounted for about the 1.0% of STb gene afterwards, gained hybrid dna is added to
In Covaris ultraphonic pipes and carry out ultrasonically treated, acquisition interrupts the piece as the 180bp close with ctDNA fragment lengths or so
Sectionization DNA.
In another implementation, also including negative quality-control product, the negative quality-control product is by following for mentioned reagent box
Mode is prepared：The EGFR wild-type cells system genomic DNA that the deletion mutation of EGFR exons 1s 9 will not contained is added to
In Covaris ultraphonic pipes and carry out ultrasonically treated, acquisition interrupts the piece as the 180bp close with ctDNA fragment lengths or so
Sectionization DNA.
A kind of PCR reaction systems for detecting the deletion mutation of human EGFR gene exons 19, including：
Wherein described primed probe mixed liquor includes thering is such as SEQ ID NO:The sense primer of sequence shown in 1, have
Such as SEQ ID NO:The anti-sense primer of sequence shown in 2, with such as SEQ ID NO:The missing of detection exons 19 of sequence shown in 3 is prominent
The fluorescence probe of change, with such as SEQ ID NO:The fluorescence probe of STb gene after the detection amplification of sequence shown in 4；Sense primer, under
The fluorescence probe of STb gene after primer, the fluorescence probe of the detection deletion mutation of exons 19 and detection are expanded is swum in PCR reaction systems
Final concentration be respectively 800nM, 800nM, 200nM and 200nM.
In another implementation, the source of the DNA profiling is extraction in tumor tissues to above-mentioned PCR reaction systems
DNA or plasma DNA, especially plasma DNA.
A kind of chip type digital pcr method for detecting the deletion mutation of human EGFR gene exons 19, comprises the following steps：
1. the reaction system of above-mentioned detection human EGFR gene exons 19 deletion mutation is prepared；
2. reaction system is loaded in digital pcr chip；
3. digital pcr chip is put into special PCR instrument, enters performing PCR amplification；
4. chip is taken out from special PCR instrument and is put to room temperature, chip is put into fluorescence letter is scanned in chip scanner
Number；
5. computer calculates mutant proportion according to fluorescence signal.
In another implementation, the program of the PCR amplifications is said chip formula digital pcr method：(1) 96 DEG C,
10min；(2) 39 circulations are proceeded, each circulation includes that 57 DEG C carry out 2min, and then 98 DEG C carry out 30sec；(3) 39
57 DEG C carry out 2min after circulation；It is kept for no more at 10 DEG C 12 hours.
The Cleaning Principle of conventional die formula digital pcr is as follows：It is nanometer level microporous containing 20,000 on digital pcr chip, use
The PCR reaction systems that Chip-Loader will be mixed are loaded onto on digital pcr chip, and mixed liquor enters because hydrophobe is acted on
Enter performing PCR reaction in the micropore of chip and in special PCR instrument.PCR reaction systems in micropore include DNA profiling, PCR amplifications
After end, wild type DNA and mutant DNA are distinguished corresponding fluorescence signal situation and can be reacted and containing the micro- of corresponding DNA profiling
Fluorescence signal after the micropore that Kong Zhong, PCR reaction system are not entered into is not expanded then is produced.Scanned with chip scanner and read
These fluorescence signal information are taken, the copy number of DNA profiling can be calculated further according to Poisson distribution principle.Because digital pcr exists
Amplification is only determined whether when carrying out result interpretation, Ct values are not relied on, the tolerance to PCR reaction suppressors is greatly improved,
Just can be with accurate quantification without necessarily referring to product and standard curve.
The design principle and conventional die formula digital pcr of the fluorescence probe of the missing of exons 19 are detected in the application kit
Difference, the fluorescence probe sequence of the detection missing of exons 19 is corresponding with the regional sequence easily lacked on exons 19.
When this design causes the missing and the mutation that any form occur when the region easily lacked on exons 19, the probe
Cannot combine, from without fluorescence signal.And detect the fluorescence probe of STb gene after amplification and be designed to not occur with exons 19
The region of missing combines, and does not have region to be crosslinked with saltant type probe, therefore no matter is dashed forward either with or without the missing that exons 19 occurs
Become, the probe can be combined and send fluorescence signal with all amplifications gained DNA.
User uses for convenience, using the mode of kit in the application still according to the behaviour of conventional die formula digital pcr
Carried out as mode, but in order that according to the method for conventional die formula digital pcr, according to fluorescence signal information and Poisson distribution principle
The actual result that the DNA profiling copy number for calculating is equally applicable in the application is, it is necessary to control herein below：Control reactant
The DNA masterplate applied sample amounts of system make the masterplate amount being distributed in each micropore on chip be 1 or 0, while the probability for 2 copies occur is non-
It is often small.If using Thermo companies3D Digital PCR System, control reaction system
DNA masterplates applied sample amount is 20-40ng, it is ensured that according to the method for conventional die formula digital pcr, according to fluorescence signal information and
The error of the actual result in DNA profiling copy number and the application that Poisson distribution principle is calculated is not more than 5%.
Compared with prior art, the present invention has the advantages that：
1. the application devises specific primer and probe according to the deletion segment of EGFR gene exons 19 so that
Detection sensitivity reaches 5/10000ths~one thousandth on Thermo QuantStudio 3D digital PCR platforms, with
ARMS-PCR is compared, and sensitivity is improved；And can also obtain sample while the yin and yang attribute of the deletion mutation of exons 19 is detected
The ratio of the mutation in this；The other detection can be detected outside 18 kinds of different types of EGFR genes simultaneously on a chip
Show sub 19 nicked forms, substantially increase versatility；The selection of targeted drug can be preferably carried out, state of illness monitoring and prognosis are commented
Estimate.
2. the digital pcr can expand the fragment less than 100bp, therefore can be not only used for the DNA that tumor tissues are extracted, and also may be used
For the amplification of ctDNA.
3. compared with drop formula digital pcr (digital droplet PCR), chip type digital pcr is without reacting PCR
System carries out droplet treatment treatment, therefore entirely the detection process time is shorter and in the absence of the inhomogenous situation of drop, ties in theory
Fruit stabilization；And cost is low compared with drop formula digital pcr, reaction flux flexibly (1~24 sample standard deviation can), drop formula digital pcr
Primary first-order equation is 8.
4. contrast is sequenced with NGS Ctdna, the time is short, speed is fast, and accuracy is high.
Brief description of the drawings
Fig. 1 is the testing result of the sample of the deletion mutation ratio 26% of EGFR gene exons 19 in the embodiment of the present invention 3；
Fig. 2 is the detection knot of the sample of the deletion mutation ratio 1.5% of EGFR gene exons 19 in the embodiment of the present invention 3
Really；
Fig. 3 is the detection knot of the sample of the deletion mutation ratio 0.1% of EGFR gene exons 19 in the embodiment of the present invention 3
Really；
Fig. 4 is the detection knot that wild-type samples of the EGFR gene without the deletion mutation of exons 19 are detected in the present invention
Really；
In Fig. 1-Fig. 4, a micropore on each point correspondence chip of display, wherein：Both no FAM fluorescence signals do not had yet
The point for having VIC fluorescence signals represents the micropore and does not enter into reaction system, therefore without any amplified signal (i.e.：Region 1)；Both
Having FAM fluorescence signals also has the point of VIC fluorescence signals (i.e.：Region 2) represent the micropore and only entered only containing wild-type template
Reaction system, the fluorescence probe for having FAM amplified signals to represent STb gene after detection is expanded normally is combined and reaction system normally expands
Increase, there are VIC amplified signals to represent exons 19 and deletion mutation do not occur, so the fluorescence probe of the detection deletion mutation of exons 19
Can normally combine and reaction system is normally expanded；There are FAM fluorescence signals but the point without VIC fluorescence signals is (i.e.：Region 3) generation
The table micropore has only entered the only reaction system containing saltant type template, has FAM amplified signals to represent the glimmering of STb gene after detection amplification
Light probe is normally combined and reaction system is normally expanded, and no VIC amplified signals represent exons 19 and deletion mutation occur, institute
Can not normally be combined with the fluorescence probe for detecting the deletion mutation of exons 19.
Specific embodiment
Below in conjunction with the accompanying drawings, specific embodiment of the invention is described in detail, it is to be understood that guarantor of the invention
Shield scope is not limited by specific embodiment.
The composition of the kit of the deletion mutation of the detection human EGFR gene of embodiment 1 exons 19
A kind of kit for detecting the deletion mutation of human EGFR gene exons 19, including：Primed probe mixed liquor sum
Word PCR reaction premixed liquids, wherein：Primed probe mixed liquor is dissolved with TE buffer solutions with such as SEQ ID NO:Shown in 1
The sense primer of sequence, with such as SEQ ID NO:The anti-sense primer of sequence shown in 2, with such as SEQ ID NO:Sequence shown in 3
Detection the deletion mutation of exons 19 fluorescence probe and with such as SEQ ID NO:STb gene after the detection amplification of sequence shown in 4
Fluorescence probe, detect that the end of fluorescence probe 5 ' of the deletion mutation of exons 19 is connected with fluorescent reporter group, be VIC, detection is outer
The end of fluorescence probe 3 ' for showing sub 19 deletion mutation is connected with quenching group, is MGB；The fluorescence probe 5 ' of STb gene after detection amplification
End is connected with fluorescent reporter group, and the fluorescent reporter group is different from the fluorescence probe of the detection deletion mutation of exons 19
Fluorescent reporter group, is FAM, and the end of fluorescence probe 3 ' of STb gene is connected with and the group of connecing is quenched after detection amplification, is MGB.Wherein：On
The concentration of trip primer and anti-sense primer in primed probe mixed liquor is 12 μM, detects that the fluorescence of the deletion mutation of exons 19 is visited
Concentration of the fluorescence probe of STb gene in primed probe mixed liquor is 3 μM after pin and detection amplification, primed probe mixed liquor
Compound method is：Will be total after sense primer, anti-sense primer, the fluorescence probe of the detection deletion mutation of exons 19 and detection amplification
Four kinds of dry powder of composition of fluorescence probe of DNA are diluted to 30 μM with TE buffer solutions respectively, then according to sense primer：Draw in downstream
Thing：Detect the fluorescence probe of the deletion mutation of exons 19：Fluorescence probe=4 of STb gene after detection amplification：4：1：1 volume ratio is mixed
Close, mixed product is primed probe mixed liquor；Digital pcr reaction premixed liquid is QuantStudioTM3D Digital
PCR Master Mix v2。
The kit of the deletion mutation of detection human EGFR gene exons 19 can also include：Positive quality control product and negative matter
Control product, positive quality control product is prepared in the following manner：By the deletion mutation cell line genomic DNA of EGFR exons 1s 9 and open country
Raw type cell line genomic DNA makes the DNA ratios of the deletion mutation of exons 1 containing EGFR gene 9 account for the pact of STb gene after mixing
1.0%, afterwards by gained hybrid dna be added in Covaris ultraphonic pipes and carry out it is ultrasonically treated, acquisition interrupt as and ctDNA
The fragmentation DNA of the close 180bp of fragment length or so；Negative quality-control product is prepared in the following manner：To not contain
The EGFR wild-type cells system genomic DNA of the deletion mutation of EGFR exons 1s 9 is added in Covaris ultraphonic pipes and carries out ultrasound
Treatment, acquisition interrupts the fragmentation DNA as the 180bp close with ctDNA fragment lengths or so.
The method of the deletion mutation of the detection human EGFR gene of embodiment 2 exons 19
1. using kit described in embodiment 1；
2. according to the form below prepares PCR reaction systems：
3., according to the specification of CHIP-LOADER, the mixed liquor of the PCR reaction systems that step 2 is prepared is loaded to number
In word pcr chip, and with mineral oil cover chip, good seal chip and check ensure without leakage；
4. chip is put into special PCR instrument, is configured according to following amplification program；
5. after amplification terminates, chip is taken out from PCR instrument, room temperature places 10min, add reading in chip scanner；
6. computer calculates mutant proportion according to fluorescence signal.
Embodiment 3 is detected using the method in embodiment 2 to standard items
Wild type standard items and mutant proportion are respectively 34%, 1.5% and 0.1% EGFR gene extron
After 19c.2236_2250del p.E746_A750del deletion mutations standard items are detected, calculated with the method in embodiment 2
Draw mutant proportion in the standard items of the 9c.2236_2250del p.E746_A750del deletion mutations of exons 1 containing EGFR gene
Respectively：34.02%th, 1.49% and 0.09%.The accuracy of detection method meets expection, its testing result such as Fig. 1-Fig. 3 institutes
Show, the testing result of wild type standard items is as shown in Figure 4.Wherein：Standard items containing mutation are obtained in the following manner：Will
EGFR mutant cells system's genomic DNA and wild-type cell system genomic DNA of known mutations ratio are mixed according to different proportion
It is then added in Covaris ultraphonic pipes and carry out ultrasonically treated after conjunction, acquisition is interrupted as close with ctDNA fragment lengths
The fragmentation DNA of 180bp or so.Wild type standard items are obtained in the following manner：EGFR exons 1s 9 will not be contained to lack
The EGFR wild-type cells system genomic DNA for losing mutation interrupts the fragment as the 180bp close with ctDNA fragment lengths or so
Change DNA.
As can be seen from Figure 3 the application kit and detection method are used, detection EGFR gene exons 19 lacks prominent
The sensitivity of change can reach 0.1%.
By the standard items only containing specific a kind of EGFR exons 1s 9 deletion mutation in table 1 below with above-mentioned same method
Detected, the accuracy for obtaining result can equally reach above standard, and sensitivity also can reach 0.1%.In addition, will be each
Standard items mixing obtains the hybrid standard product containing the deletion mutation of following 18 kinds of EGFR exons 1s 9 simultaneously, with above-mentioned same side
Method is detected that the accuracy for obtaining result can equally reach above standard, and sensitivity also can reach 0.1%.
Table 1
| c.2235_2249del p.E746_A750del |
| c.2236_2250del p.E746_A750del |
| c.2236_2253del p.E746_T751del |
| c.2237_2251del p.E746_T751>A |
| c.2237_2254del p.E746_S752>A |
| c.2238_2255del p.E746_S752>D |
| c.2239_2247del p.L747_E749del |
| c.2239_2253del p.L747_T751del |
| c.2239_2256del p.L747_S752del |
| c.2240_2251del p.L747_T751>S |
| c.2240_2254del p.L747_T751del |
| c.2240_2257del p.L747_P753>S |
| c.2235_2252>AAT(complex)p.E746_T751>I |
| c.2237_2250>T(complex)p.E746_S752>V |
| c.2238_2248>GC(complex)p.L747_A750>P |
| c.2238_2252>GCA(complex)p.L747_T751>Q |
| c.2239_2248TTAAGAGAAG>C(complex)p.L747_A750>P |
| c.2239_2251>C(complex):p.L747_T751>P |
Standard items in table 1 can be bought by commercial channel and be obtained
Embodiment 4 is detected using the method in embodiment 2 to actual sample
To 20 clinics be using the method in embodiment 2 3~4 phase non-small cell lung cancer patients blood sample and with blood sample pair
The tissue samples answered are detected.
First, in blood sample ctDNA detection：
1. mix the whole blood sample 10ml (sample collection be no more than 3 hours) that is stored in EDTA anticoagulant tubes and be transferred to
In 15ml centrifuge tubes, 2000g, 4 DEG C of centrifugation 10min take the blood plasma of clarification；
2. blood plasma is dispensed into 1.5ml pipes, setting rotating speed is that 16000g is centrifuged 15min in being positioned over 4 DEG C of centrifuges, is taken
Clarification blood plasma；
3. with the ctDNA extracts kits (MagMAX of Thermo Fisher Scientific companiesTMCell-Free
DNA Isolation Kit), ctDNA is extracted in operation to specifications；
4.ctDNA confirms that total amount is no less than 20ng after being quantified through quality inspection and Qubit3.0, according to the detection side in embodiment 2
Method is detected.
2nd, the detection of the genomic DNA in tissue samples：
20 tissue samples corresponding with blood sample detect with EGFR mutation detection kits (ARMS methods), the sun of discovery
Property sample continue with sanger be sequenced verify, as a result unanimously.
3rd, testing result is as follows：
Result shows that 20 tissue detections of sample are completely the same with ctDNA testing results, illustrates that this kit reaches
Extraordinary effect.
The foregoing description to specific illustrative embodiment of the invention be in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to above-mentioned teaching, can be much changed
And change.The purpose of selecting and describing the exemplary embodiment is that explaining that certain principles of the invention and its reality should
With so that those skilled in the art can realize and using a variety of exemplaries of the invention and
A variety of selections and change.The scope of the present invention is intended to be limited by claims and its equivalents.
Claims (10)
1. a kind of kit for detecting the deletion mutation of human EGFR gene exons 19, it is characterised in that including：With such as SEQ
ID NO:The sense primer of sequence shown in 1, with such as SEQ ID NO:The anti-sense primer of sequence shown in 2, with such as SEQ ID
NO:Sequence shown in 3 detection the deletion mutation of exons 19 fluorescence probe and with such as SEQ ID NO:The detection of sequence shown in 4
The fluorescence probe of STb gene after amplification.
2. kit according to claim 1, it is characterised in that the sense primer, anti-sense primer, detection exons 19
The fluorescence probe of STb gene is present in primed probe mixed liquor after the fluorescence probe of deletion mutation and detection amplification.
3. kit according to claim 2, it is characterised in that the sense primer is dense in primed probe mixed liquor
It is 12 μM to spend, and concentration of the anti-sense primer in primed probe mixed liquor is 12 μM, the detection deletion mutation of exons 19
Concentration of the fluorescence probe in primed probe mixed liquor be 3 μM, the fluorescence probe of STb gene is visited in primer after the detection amplification
Concentration in pin mixed liquor is 3 μM.
4. kit according to claim 1, it is characterised in that also including digital pcr reaction premixed liquid, preferably
QuantStudioTM3D Digital PCR Master Mix v2。
5. kit according to claim 1, it is characterised in that also including positive quality control product, the positive quality control product is led to
In the following manner is crossed to prepare：By the deletion mutation cell line genomic DNA of EGFR gene exons 19 and wild-type cell system base
Because making the DNA ratios of the deletion mutation of exons 1 containing EGFR gene 9 account for about the 1.0% of STb gene after group DNA mixing, gained is mixed
DNA is added in Covaris ultraphonic pipes and carries out ultrasonically treated, and acquisition interrupts left as the 180bp close with ctDNA fragment lengths
Right fragmentation DNA.
6. kit according to claim 1, it is characterised in that also including negative quality-control product, the negative quality-control product leads to
In the following manner is crossed to prepare：The EGFR wild-type cells system genomic DNA that the deletion mutation of EGFR exons 1s 9 will not contained adds
To in Covaris ultraphonic pipes and carry out ultrasonically treated, acquisition is interrupted as the 180bp close with ctDNA fragment lengths or so
Fragmentation DNA.
7. a kind of PCR reaction systems for detecting the deletion mutation of human EGFR gene exons 19, it is characterised in that including：
8. PCR reaction systems according to claim 6, it is characterised in that during the source of the DNA profiling is tumor tissues
The DNA or plasma DNA of extraction, especially plasma DNA.
9. a kind of chip type digital pcr method for detecting the deletion mutation of human EGFR gene exons 19, it is characterised in that including
Following steps：
1) the PCR reaction systems of the deletion mutation of detection human EGFR gene exons 19 described in claim 7 are prepared；
2) reaction system is loaded in digital pcr chip；
3) digital pcr chip is put into special PCR instrument, enters performing PCR amplification；
4) chip is taken out from special PCR instrument and is put to room temperature, chip is put into chip scanner and scans fluorescence signal；
5) computer calculates mutant proportion according to fluorescence signal.
10. chip type digital pcr method according to claim 9, it is characterised in that the program of the PCR amplifications is：
(1) 96 DEG C, 10min；(2) 39 circulations are proceeded, each circulation includes that 57 DEG C carry out 2min, and then 98 DEG C carry out 30sec；
2min is carried out at 57 DEG C after (3) 39 circulations, is kept for no more 12 hours at 10 DEG C afterwards.
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