CN110373467A - A kind of detection reagent and detection method of human EGFR gene mutations site T790M - Google Patents
A kind of detection reagent and detection method of human EGFR gene mutations site T790M Download PDFInfo
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Abstract
A kind of detection reagent and detection method of human EGFR gene mutations site T790M, the detection reagent include: PCR reaction solution I, PCR reaction solution II, enzymic digestion reaction solution, Single base extension liquid I, Single base extension liquid II and magnetic particle suspension；Detection method based on the reagent includes PCR reaction solution I, PCR reaction solution II expands the EGFR gene in blood sample, and the digestion of enzymic digestion reaction solution is added, Single base extension liquid I is added in the product of digestion, Single base extension liquid II and PCR-SAP product carries out Single base extension, carries out chip analysis after product enrichment with magnetic bead.It can detect that sensitivity reaches 0.1% down to 10 mutant DNAs from 10^4 DNA molecular provided by the present invention for the detection reagent and method of detection human EGFR gene p.T790M mutation, realize the trace detection of T790M.
Description
Technical field
The present invention relates to human EGFR gene mutations site p.T790M (c.2369C > T) detection technique fields, and in particular to
PCR technology and single base extension realize the trace detection of p.T790M in conjunction with magnetic particle technology.
Background technique
EGFR is a kind of glycoprotein receptor of cell membrane surface, has tyrosine kinase (Tyrosine Kinase, TK) living
Property, it is the expression product of proto-oncogene c-erbB-1 (HER-1).The key signal transduction approach of EGFR has: PI3K-PDK is logical
Road, RAS-RAF-MEK-ERK-MAPK access, PLC- γ access, JAK-STAT access.By these approach, by extracellular signal
It is converted into intracellular signal, to successfully manage extraneous signal stimulus, growth, proliferation, the differentiation of cell is adjusted, inhibits cell
Apoptosis.
U.S. Food and Drug Administration (FDA) had approved AstraZeneca (Astrazeneca) company in 2003
EGFR small molecule targeted drug Gefitinib (Gefitinib, it may be assumed that Iressa/Iressa) is for treating Advanced Non-Small Cell lung
Cancer (NSCLC), China had approved Gefitinib in 2005 and enter clinical treatment.The invertibity tyrosine special as EGFR
Kinase inhibitor (EGFR-TKI), Gefitinib by with ATP competitive binding tyrosine kinase, play its inhibiting effect.
With being widely used for Gefitinib, there is drug resistance after treating 1-2 in the patient of about 50-65%, main
Molecular mechanism is the appearance of acquired resistance mutation EGFR p.T790M (c.2369C > T).The site is located at EGFR tyrosine-kinase
The bond area ATP of enzyme after amino acid changes into M (methionine) by T (threonine), then reduces the affine of the region and ATP
Property, to reduce the validity of drug.Therefore, p.T790M is found before medication and as early as possible (c.2369C in drug use process
> T) medicament-resistant mutation, there is important clinical meaning.
Currently, the detection method for EGFR p.T790M gene mutation is more, sensitivity 29-71%, specificity is
67-100%.Mainly there are Super ARMS-PCR method, drop digital pcr for minigene mutation detection methods
The side such as (droplet digital PCR, ddPCR), high-flux sequence (Next-Generation Sequencing, NGS)
Method.The mutation down to 0.1%, but the former can be detected in Super ARMS PCR sensitivity about 0.2-0.8%, ddPCR and NGS
There are false positive results, detection threshold values to be not easy the disadvantages of determining, the latter analyzes instrument and equipment, reviewer and data and requires
It is higher, the disadvantages of period is longer, costly, all it is not easy to clinical expansion.
Therefore, if micro EGFR p.T790M quick and precisely can delicately be detected under a large amount of normal DNA backgrounds
(c.2369C > T) mutation strives for that therapic opportunity has important meaning for the therapeutic strategy of clinical EGFR targeted drug for patient
Justice.
Summary of the invention
Technical problem is as follows:
In a first aspect, quick and precisely can delicately be detected micro- under a large amount of normal DNA backgrounds the present invention provides a kind of
The method of human EGFR p.T790M (c.2369C > T) mutation of amount.
Second aspect, the present invention provides the detection reagents of human EGFR gene mutations site T790M a kind of.
Technical solution is as follows:
A kind of detection reagent of human EGFR gene mutations site T790M, the detection reagent include:
PCR reaction solution I；
PCR reaction solution II；
Enzymic digestion reaction solution；
Single base extension liquid I；
Single base extension liquid II；
Magnetic particle suspension；
Wherein, the PCR reaction solution I includes the amplification upstream and downstream primer and its change of EGFR genetic mutation site T790M
Body；
Wherein, the PCR reaction solution II includes archaeal dna polymerase, dNTP, buffer and Mg2+ needed for PCR reaction；
Wherein, the enzymic digestion reaction solution can digest primer sequence and dNTPs in reaction system；
Wherein, the Single base extension liquid I includes the extension primer and its variant of EGFR genetic mutation site T790M；
Wherein, the Single base extension liquid II includes iPLEX enzyme, biotin labeling needed for single base extension
DdT and buffer；
Wherein, the magnetic particle in the magnetic particle suspension marks Avidin.
The amplification upstream and downstream primer and its variant using EGFR c.2369C > the upstream and downstream sequence of T designs as target sequence,
It can be using EGFR c.2369C > upstream and downstream at most 200bp, 150bp, 100bp, 75bp, 50bp, 20bp of T is target sequence
Design.
The amplification upstream and downstream primer and its variant using EGFR c.2369C > the upstream and downstream 75bp of T designs as target sequence.
The amplification upstream and downstream primer and its variant include: the complementary series of the appended sequence at 5 ' ends and 3 ' ends；Institute
The appended sequence stated cannot match complementary completely with template sequence or can partially match complementation；The appended sequence is for mentioning
The molecular weight of high entire PCR primer, to distinguish extension products；The complementary series and EGFR DNA complete complementary.
The appended sequence length is 10bp；The nonvolatile memory of the amplification upstream and downstream primer is 18bp-
27bp, G/C content 40-60%, BLAST detection do not have complementarity with other genes existing for human plasma, itself is not easy shape
At serious hairpin structure and dimeric structure.
The appended sequence of the amplification upstream and downstream primer is equal are as follows:
Appended sequence: 5 '-ACGTTGGATG-3 '；
The complementary series of the amplification upstream and downstream primer is respectively as follows:
The complementary series of upstream primer: 5 '-ATCTGCCTCACCTCCACC-3 '；
The complementary series of downstream primer: 5 '-TGTTCCCGGACATAGTCC-3 '；
The amplification upstream and downstream primer are as follows:
Upstream primer: 5 '-ACGTTGGATGATCTGCCTCACCTCCACC-3 '；
Downstream primer: 5 '-ACGTTGGATGTGTTCCCGGACATAGTCC-3 '.
The archaeal dna polymerase is a kind of archaeal dna polymerase resistant to high temperature dependent on DNA profiling.
The archaeal dna polymerase can be selected from Taq archaeal dna polymerase, high-fidelity DNA polymerase, thermal starting archaeal dna polymerase,
Taq Plus archaeal dna polymerase, Long Taq archaeal dna polymerase, any one in direct DNA amplification polymerase.
The archaeal dna polymerase is selected from Taq archaeal dna polymerase.
The enzymic digestion reaction solution includes shrimp alkaline phosphotase (SAP).
The extension primer and its variant is complementary with the sequence of mutational site upstream, 3 ' end last bit bases just with
EGFR c.2369C > base pair complementarity of the upstream T first.
The extension primer and its end of variant 3 ' are complementary with the sequence of mutational site upstream including at least 10 bases,
It can be at least ten, 15,20,25,30 base pair complementarities, 5 ' ends can add protection base and/or attached
Add base, the 5 ' ends can be with or without the sequence complementation of mutational site upstream.
The extension primer are as follows:
Extension primer: 5 '-CGTGCAGCTCATCA-3 '.
A kind of detection method of human EGFR gene mutations site p.T790M, this method can be to blood or bodily tissues
Middle EGFR gene mutation site p.T790M is detected, and this method recycles extension primer special by PCR amplification target fragment
Specific amplification mutated gene improves accuracy and sensitivity after further magnetic particle technology enrichment.
Method includes the following steps:
(1) sample preparation: at paraffin-embedded tissue, flesh tissue, frost investing tissue, blood, blood plasma and hydrothorax routine
Reason extracts DNA sample；
(2) target fragment PCR amplification: PCR reaction solution I, PCR reaction solution II being added into reaction tube, and DNA sample is prepared
PCR reaction system；PCR reaction condition is arranged in PCR instrument, carries out PCR；
(3) enzymic digestion: PCR reaction product is added and is mixed with enzymic digestion reaction solution, is reacted under certain condition, reaction is completed
Afterwards, the inactivation of enzyme is carried out；
(4) Single base extension liquid I, Single base extension liquid II, PCR-SAP product Single base extension: are added into reaction tube
Mixing；PCR reaction condition is arranged in PCR instrument, carries out Single base extension；
(5) enrichment with magnetic bead reacts: magnetic particle suspension is added in range by a certain percentage and extension products mix and first time
It is incubated for, product, which is placed on magnetic frame after magnetic bead adsorbs completely, discards supernatant liquid, and cleans magnetic bead with HPLC- water, and magnetic bead is resuspended
It is incubated for for second in biotin coating buffer, carries out desalination after rear of short duration centrifugation；
(6) point sample is analyzed with data: after the sample point sample after desalination is mixed to CPM chip with matrix, carrying out Mass Spectrometer Method
And data are analyzed.
The PCR instrument can be selected from common PCR instrument, grads PCR instrument, In situPCR instrument, real-time fluorescence quantitative PCR instrument
In any one.
The PCR instrument is common PCR instrument.
The mode mixed in the step 5) can be selected from and be mixed by inversion, manual and/or machine；Rotation mixes, manually
And/or machine；Space or horizontal direction shake, any one manually and/or in machine or combinations thereof；The step 4)
The program of middle mixing stage can mix, or continuous mixing.
The mode of the mixing is machine mixing
The machine of the mixing can be selected from rotary mixer, roller mixer, rail mounted shaking table, wraping plate shaking table, examination
Pipe oscillator, rotary incubator, eddy mixer, micro oscillator, any one in Mute mixer.
The machine of the mixing is rotary mixer；The program of the mixing is continuous mixes.
The temperature being incubated for for the first time in the step 5) can selected from any one in 42 DEG C, 37 DEG C, room temperature, 4 DEG C,
The time that the first time is incubated for matches with incubation temperature, reduces with temperature, and incubation time increases, can be selected from least
For 5min, 15min, 30min, stay overnight.
The temperature being incubated for for the first time in the step 5) is room temperature, incubation time 30min.
The temperature that second is incubated in the step 5) is 90 DEG C, incubation time 5min.
It is that desalination is carried out by resin in the step 5).
It has the beneficial effect that:
Detection reagent and method provided by the present invention for detection human EGFR gene p.T790M mutation can be from 10^4
Detect that down to 10 mutant DNAs, sensitivity reaches 0.1%, realizes the trace detection of p.T790M in DNA molecule.
Detailed description of the invention
Fig. 1 is EGFR p.T790M abrupt climatic change schematic illustration provided by the invention.
Fig. 2 is sensitivity technique of the invention, and mutated gene copy number is total copy number respectively in cell strain double-stranded DNA
5%, 2 %, 1%, 0.5%, 0.2%, 0.1% testing result.
Fig. 3 is sensitivity technique of the invention, and mutated gene copy number is total copy number in respectively synthesizing single-stranded DNA
5%, 2%, 1%, 0.5%, 0.2%, 0.1% testing result.
Fig. 4 is specific detection of the invention, respectively without mutated gene copy number in DNA sample, cell strain double-stranded DNA
For 0%, the 80% of total copy number and the testing result of eluent (magnetic bead adsorption reaction buffer), A is no DNA in attached drawing
Sample；B is 0% that mutated gene copy number is total copy number in attached drawing；It is always to copy that C, which is mutated gene copy number, in attached drawing
Several 80%；D is eluent (magnetic bead adsorption reaction buffer) in attached drawing.
Specific embodiment
With reference to attached drawing to the present invention a kind of detection reagent and detection method of human EGFR gene mutations site p.T790M,
It is described in detail.
The present invention is the detection reagent and its detection method of a kind of human EGFR gene mutations site p.T790M, the detection
Reagent and detection method can be used for detecting EGFR genetic mutation site T790M in blood or bodily tissue.Preferably
Blood, the detection of blood plasma and hydrothorax sample；But its application is moreover, can be also used for the detection of tissue sample.It is examined
Test sample originally can be selected from paraffin-embedded tissue, and flesh tissue freezes investing tissue, blood, blood plasma and hydrothorax etc., the inspection of acquisition
This need to can carry out DNA extraction to test sample by conventional treatment, and DNA extraction can select commercially available nucleic acid extraction kit or root
According to " modern molecular biology technique and experiment skill " leaf chess, dense chief editor is write, and carries out DNA extraction.
The present invention is the detection reagent of human EGFR gene mutations site p.T790M a kind of, which includes: PCR
Reaction solution I；PCR reaction solution II；Enzymic digestion reaction solution；Single base extension liquid I；Single base extension liquid II；Magnetic particle suspension；
Wherein, the PCR reaction solution I includes the amplification upstream and downstream primer and its variant of EGFR genetic mutation site p.T790M；Its
In, the PCR reaction solution II includes archaeal dna polymerase, dNTP, buffer and Mg2+ needed for PCR reaction；Wherein, described
Enzymic digestion reaction solution can digest primer sequence and dNTPs in reaction system；Wherein, the Single base extension liquid I packet
The extension primer and its variant of the p.T790M of site containing EGFR genetic mutation；Wherein, the Single base extension liquid II includes single
IPLEX enzyme needed for base extension, biotin labeling ddT and buffer；Wherein, in the magnetic particle suspension
Magnetic particle marks Avidin.
In order to accurately sensitively expand to the sequence of the p.T790M of site containing EGFR genetic mutation, used draws
Object sequence must design rationally, rationally include: amplification upstream and downstream primer and its variant with EGFR p.T790M (c.2369C >
T upstream and downstream sequence) designs for target sequence, can be with EGFR c.2369C > upstream and downstream at most 200bp, 150bp of T,
100bp, 75bp, 50bp, 20bp design for target sequence.Its principle designed allows for the EGFR gene piece in blood sample
Duan great little is typically small, in 150bp or so.
Preferably, expand upstream and downstream primer and its variant using EGFR c.2369C > the upstream and downstream 75bp of T sets as target sequence
Meter；Its specific location is obtained by experiment screening, the too long or too short detection for being all unfavorable for target gene in blood sample.
In order to accurately sensitively be expanded to the sequence of the T790M of site containing EGFR genetic mutation, primer used
Sequence must design rationally, rationally further include: amplification upstream and downstream primer must be distinguished with extension products, must satisfy simultaneously
PCR primer designs basic principle, including primer length (primer length), and G/C content, Tm value, itself avoids complementation,
Specificity etc..In the present invention, amplification upstream and downstream primer includes the appended sequence at 5 ' ends and the complementary series at 3 ' ends；Described is attached
Add sequence that cannot match complementary completely with template sequence or can partially match complementation；The appended sequence is entire for improving
The molecular weight of PCR primer, to distinguish extension products；The complementary series and EGFR DNA complete complementary.
Preferably, appended sequence length is 10bp；The nonvolatile memory for expanding upstream and downstream primer is 18bp-27bp, GC
Content is 40-60%, and BLAST detection does not have complementarity with other genes existing for human plasma, itself is not easy to be formed serious
Hairpin structure and dimeric structure.
Preferably, the appended sequence for expanding upstream and downstream primer is equal are as follows: appended sequence: 5 '-ACGTTGGATG-3 '；
The complementary series of the amplification upstream and downstream primer is respectively as follows:
Upstream primer complementary series: 5 '-ATCTGCCTCACCTCCACC-3 '；
Downstream primer complementary series: 5 '-TGTTCCCGGACATAGTCC-3 '；
The amplification upstream and downstream primer are as follows:
Upstream primer: 5 '-ACGTTGGATGATCTGCCTCACCTCCACC-3 '；
Downstream primer: 5 '-ACGTTGGATGTGTTCCCGGACATAGTCC-3 '.
The primer sequence is all that screening obtains, and has optimal accuracy and PCR effect.
In order to make PCR effect best, archaeal dna polymerase used in PCR of the present invention is a kind of resistant to high temperature dependent on DNA profiling
Archaeal dna polymerase.It can be Taq archaeal dna polymerase, high-fidelity DNA polymerase, thermal starting archaeal dna polymerase, Taq Plus
Archaeal dna polymerase, Long Taq archaeal dna polymerase, any one in direct DNA amplification polymerase；Itself and T4DNA polymerase,
The Klenow segment of e. coli dna polymerase 1 is compared, it is easier to realize that PCR operating process automates.
Preferably, thermal starting Taq archaeal dna polymerase, is usually used in Standard PCR.
In order to carry out single base extension, while guaranteeing that the base extended contains only ddT, it is necessary to carry out enzymic digestion reaction
Hydrolyze remaining dNTPs in PCR reaction system.
Preferably, the enzymic digestion reaction solution includes shrimp alkaline phosphotase (SAP), is catalyzed the base of phosphorous acid compound
Hydrolysis.
In pcr amplification reaction, upstream primer and downstream primer expand to obtain target fragment, containing wild type and saltant type
DNA profiling is expanded simultaneously.In extension, extension primer only occurs using target fragment as template in mutating alkali yl position
Single base extension.Since in extension system, only the ddT of addition saltant type, is not added the ddC of matching wild type DNA,
Therefore extension products are only variants.Therefore, extension primer and its variant are complementary with the sequence of mutational site upstream, and 3 '
Hold last bit base just with EGFR c.2369C > base pair complementarity of the upstream T first.
Preferably, extension primer and its end of variant 3 ' are complementary with the sequence of mutational site upstream including at least 10 bases,
It can be at least ten, 15,20,25,30 base pair complementarities, 5 ' ends can add protection base and/or
Additional base, 5 ' ends can be with or without the sequence complementations of mutational site upstream.Its design principle is that primer conventional design is former
Then.
Preferably, extension primer: 5 '-CGTGCAGCTCATCA-3 '.
A kind of detection method of human EGFR gene mutations site T790M, method includes the following steps:
1) sample preparation: paraffin-embedded tissue, flesh tissue freeze investing tissue, blood, blood plasma and hydrothorax conventional treatment
Extract DNA sample；
2) target fragment PCR amplification: PCR reaction solution I, PCR reaction solution II being added into reaction tube, and DNA sample is prepared
PCR reaction system；PCR reaction condition is arranged in PCR instrument, carries out PCR；
3) enzymic digestion: being added PCR reaction product and mix with enzymic digestion reaction solution, react under certain condition, after the reaction was completed,
Carry out the inactivation of enzyme；
4) Single base extension: Single base extension liquid I being added into reaction tube, and Single base extension liquid II, PCR-SAP product is mixed
It closes；PCR reaction condition is arranged in PCR instrument, carries out Single base extension；
5) enrichment with magnetic bead reacts: magnetic particle suspension is added in range by a certain percentage and extension products are mixed and incubated for the first time
It educates, product, which is placed on magnetic frame after magnetic bead adsorbs completely, discards supernatant liquid, and cleans magnetic bead with HPLC- water, and magnetic bead is resuspended in
It is incubated for for second in biotin coating buffer, carries out desalination after rear of short duration centrifugation；
6) point sample is analyzed with data: after the sample point sample after desalination is mixed to CPM chip with matrix, by MassARRAY
The detection of 4 mass spectrograph of Analyzer carries out data analysis by TyperAnalyzer software.
In above entire reaction, PCR instrument is conventional PCR instrument, but its is unlimited in this way, it can also select band function
Can property PCR instrument, such as: grads PCR instrument, In situPCR instrument, any one in real-time fluorescence quantitative PCR instrument.
Preferably, common PCR instrument.
The influence factor of enrichment with magnetic bead reaction is numerous comprising the mixing of magnetic particle suspension and extension products and incubation side
Formula, in the present invention, the mode of mixing can be selected from being mixed by inversion, manually and/or machine；Rotation mixes, manual and/or machine；
Space or horizontal direction shake, any one manually and/or in machine or combinations thereof；The program of mixing can be with stage mixed
It is even, or continuous mixing.
Preferably machine mixes, and advantage is that repeatability is higher.
In the present invention, the machine of mixing can selected from rotary mixer, roller mixer, rail mounted shaking table, wraping plate shaking table,
Test tube oscillator, rotary incubator, eddy mixer, micro oscillator, any one in Mute mixer.But it selects
Different and its mixing, effect have difference, can be with optimum selecting according to laboratory facility.In the present invention, from magnetic particle
The centrifuge tube size and suspension state that suspension itself loads are set out, preferably rotary mixer, and mixing program is continuous mix.
Equally, in the influence factor of enrichment with magnetic bead reaction, incubation temperature and time have great influence；It is incubated for for the first time
Temperature can be selected from any one in 42 DEG C, 37 DEG C, room temperature, 4 DEG C, and the time being incubated for for the first time matches with incubation temperature, with
Temperature reduces, and incubation time increases, and can 15min, 30min, stay overnight selected from 5min is at least.
Preferably, the temperature of incubation is room temperature, incubation time 30min.Its reaction condition is that experiment is groped, and reacts item
Part is related to biotin-avidin system, and since the system research is more mature, the reaction condition in the present invention is not
As unique conditional, can suitably adjust.
Second of temperature being incubated for is 90 DEG C, incubation time 5min, and reaction condition is that experiment is groped.
It further includes desalting steps, and method is that resin is added.
Embodiment 1
The preparation of 1.PCR reaction solution I and Single base extension liquid I
It is prominent with EGFR p.T790M according to the EGFR gene wild-type sequence (NC_000007.13) that ncbi database is announced
Displacement point upstream and downstream 75bp is reference, designs specificity EGFR amplimer；Mutational site EGFR (c.2369C > T).
PCR amplification primer length is 28 bases, and 5 ' ends increase ACGTTGGATG totally 10 bases, improve entire PCR and draw
The molecular weight of object, to distinguish extension products, 18 bases then with EGFR DNA complete complementary in sample to be tested.
Extension products sequence is located in PCR product sequence, complementary with the sequence of mutational site upstream.
For detecting the primer sequence of human EGFR gene p.T790M mutation:
PCR amplification upstream primer: 5 '-ACGTTGGATGATCTGCCTCACCTCCACC-3 '；
PCR amplification downstream primer: 5 '-ACGTTGGATGTGTTCCCGGACATAGTCC-3 '；
Extension primer: 5 '-CGTGCAGCTCATCA-3 '.
PCR reaction solution I are as follows: buffer or water and amplification upstream and downstream primer are configured to PCR primer mix (1.0 μM).
Single base extension liquid I are as follows: buffer or water and extension primer are configured to extension primer (10uM).
The preparation of 2.PCR reaction solution II and Single base extension liquid II
PCR reaction solution II and Single base extension liquid II is the solution that primer is free of in PCR reaction system, the solution packet
Contain: archaeal dna polymerase, substrate (dNTP), buffer, HPLC- water；Various composition needed for it can be from commercially available (such as NEB)
Purchase.This laboratory uses commercially available (Qiagen Hotstar Taq kit) and (Agena iPLEX pro according to self-condition
kit)。
Unit volume (4.5 μ L-18 μ L) PCR reaction solution II includes:
| PCR reaction solution II | Dosage (μ L) |
| 10x PCR buffer | 2.0 |
| MgCl2(25mM) | 1.6 |
| dNTP(25mM) | 0.4 |
| HotStar Taq enzyme (5U/ μ L) | 0.5(2.5U) |
| HPLC- water | 0-13.5 |
Unit volume (1 μ L) Single base extension liquid II includes:
| Single base extension liquid II | Dosage (μ L) |
| 10x iPLEX Pro Buffer | 0.20 |
| IPLEX Pro enzyme | 0.04 |
| Terminating nucleotide ddT (1mM) | 0.05 |
| HPLC- water | 0.71 |
3. the preparation of enzymic digestion reaction solution
Unit volume (2 μ L) enzymic digestion reaction solution includes:
| Reagent (Agena) | Dosage (μ L) |
| SAP buffer | 0.17 |
| SAP enzyme | 0.30 |
| HPLC- water | 1.53 |
4. the preparation of magnetic particle suspension
Unit volume (41 μ L) magnetic particle (Agena) suspension configuration method are as follows: take 4.25 μ L magnetic beads through binding and
After wash buffer is handled 2 times, 25 μ L binding and wash buffer are resuspended in, 16 μ L HPLC- water are added.
Embodiment 2
1. the detection method of human EGFR gene mutations site T790M
EGFR p.T790M abrupt climatic change schematic illustration such as Fig. 1.
1) sample preparation: paraffin-embedded tissue, flesh tissue freeze investing tissue, blood, blood plasma and hydrothorax conventional treatment
Extract DNA sample.
2) target fragment PCR amplification: PCR reaction solution I, PCR reaction solution II being added into reaction tube, and DNA sample is prepared
PCR reaction system；PCR reaction condition is arranged in PCR instrument, carries out PCR.The dosage of component prepares PCR reaction system according to the form below
(20 μ L): DNA sample is added according to actual requirement, volume is 2-10 μ L, and is mended with water to 20 μ L.
PCR reaction condition: 95 DEG C of initial denaturation 15min；95 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 60s are carried out
45 circulations；72 DEG C of final extension 5min.
3) enzymic digestion: totally 7 μ L at 37 ° react 40min after taking 2 μ L of enzymic digestion reaction solution and 5 μ L PCR products to mix, in 85
DEG C inactivation 5min.
4) Single base extension: the dosage of component prepares PCR reaction system (9 μ L) according to the form below.
| Detection reagent | Dosage (μ L) |
| Single base extension liquid I (10uM) | 1 |
| PCR-SAP product | 7 |
| Single base extension liquid II | 1 |
Single base extension condition: 94 DEG C of 30s；94 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s, totally 40 recycle, wherein often
52 DEG C of 5s, 80 DEG C of 5s are repeated 5 times in a circulation；72 DEG C of extension 3min.
5) enrichment with magnetic bead react: after being mixed with 9 μ L extension products with 41 μ L magnetic particle suspensions on rotary mixer in
Mixed at room temperature 30min.50 μ L products, which are placed on magnetic frame after magnetic bead adsorbs completely, discards supernatant liquid, and with 100 μ L HPLC-
Water cleans magnetic bead 2 times.Finally, magnetic bead is resuspended in 13 μ L magnetic bead eluents (agena), the of short duration centrifugation after 90 DEG C of heating 5min
After be added 2 μ L resins carry out desalination.
6) point sample is analyzed with data: after about 30nL point sample is mixed to CPM chip with matrix, by MassARRAY Analyzer
The detection of 4 mass spectrographs carries out data analysis by TyperAnalyzer software.
2. the sensitivity evaluation of detection method:
Material: the cell strain NCI-H1975 in the mutational site EGFR p.T790M, purchased from Shun, slowly (Shanghai) biotechnology is limited
Company；Wild-type cell strain A549, purchased from Ran Shun (Shanghai) Biotechnology Co., Ltd.
1) DNA sample tests the sensitivity of party's science of law:
To the NCI-H1975 cell strain DNA being mutated containing EGFR p.T790M, use A549 cell strain DNA as wild type
DNA。
Cell strain is in 1640 culture medium of RPMI (Gibco) routine culture for containing 10% fetal calf serum (Gibco).Wait train
It after feeding ware covers with, is digested with 0.25%Trypsin-EDTA (Gibco), and is resuspended in 200ul PBS according to QIAamp
DNA Mini Kit specification carries out DNA extraction.
Concentration is quantitatively calculated by picogreen and is mixed, and be mutated gene copy number is total copy number (10000 copy)
5%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0% detected, the concentration of DNA is 33ng after mixing, and experimental result is such as
Fig. 2, the peak height of 0.1% abrupt climatic change detail as the result is shown, shows to can detecte the mutation down to 0.1%.
The EGFR p.T790M mutated gene of the fragmentation according to present in blood sample, further amplification synthesizes wild type and dashes forward
Modification single stranded DNA calculates concentration by measurement absorbance, is respectively that (- 10000 copy total copy number with mutated gene copy number
Shellfish) 5%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0% detected, experimental result such as Fig. 3,0.1% abrupt climatic change
The peak height of detail as the result is shown shows to can detecte the mutation down to 0.1%.
3. the Evaluation on specificity of detection method:
It is carried out respectively using A549 cell strain and the known NCI-H1975 cell strain DNA for containing p.T790M mutation as template
Detection, to assess the specificity of this method.As a result as shown in figure 4, wherein for A without DNA sample, B is 0% gene mutation sample, C
For 80% mutant DNA template, D is the buffer of enrichment with magnetic bead reaction.The result shows that provided by the present invention for detecting the mankind
The method of EGFR gene p.T790M mutation can specific detection EGFR gene p.T790M (c.2369C > T) site mutation.
In conclusion the site that the method provided by the present invention can detect human EGFR gene p.T790M (c.2369C > T) is prominent
Become, sensitivity with higher and specificity, the detection method for human EGFR gene p.T790M mutation provides a kind of selection.
Claims (9)
1. a kind of detection reagent of human EGFR gene mutations site T790M, which is characterized in that the detection reagent includes:
PCR reaction solution I；
PCR reaction solution II；
Enzymic digestion reaction solution；
Single base extension liquid I；
Single base extension liquid II；
Magnetic particle suspension；
Wherein, the PCR reaction solution I includes the amplification upstream and downstream primer and its variant of EGFR genetic mutation site T790M；
Wherein, the PCR reaction solution II includes archaeal dna polymerase, dNTP, buffer needed for PCR reaction；
Wherein, the enzymic digestion reaction solution can digest the dNTPs in reaction system；
Wherein, the Single base extension liquid I includes the extension primer and its variant of EGFR genetic mutation site T790M；
Wherein, the Single base extension liquid II include single base extension needed for iPLEX enzyme, biotin labeling ddT and
buffer；
Wherein, the magnetic particle in the magnetic particle suspension marks Avidin.
2. the detection reagent of human EGFR gene mutations site T790M according to claim 1 a kind of, which is characterized in that
The amplification upstream and downstream primer and its variant using EGFR c.2369C > the upstream and downstream 75bp of T designs as target sequence.
3. the detection reagent of human EGFR gene mutations site T790M according to claim 1 a kind of, which is characterized in that
The amplification upstream and downstream primer and its variant include: the complementary series of the appended sequence at 5 ' ends and 3 ' ends；Described is additional
Sequence cannot match complementary completely with template sequence or can partially match complementation；The appended sequence is for improving entire PCR
The molecular weight of primer, to distinguish extension products；The complementary series and EGFR DNA complete complementary.
4. the detection reagent of human EGFR gene mutations site T790M according to claim 3 a kind of, which is characterized in that
The appended sequence of the amplification upstream and downstream primer is equal are as follows:
Appended sequence: 5 '-ACGTTGGATG-3 '；
The complementary series of the amplification upstream and downstream primer is respectively as follows:
The complementary series of upstream primer: 5 '-ATCTGCCTCACCTCCACC-3 '；
The complementary series of downstream primer: 5 '-TGTTCCCGGACATAGTCC-3 '；
The amplification upstream and downstream primer are as follows:
Upstream primer: 5 '-ACGTTGGATGATCTGCCTCACCTCCACC-3 '；
Downstream primer: 5 '-ACGTTGGATGTGTTCCCGGACATAGTCC-3 '.
5. the detection reagent of human EGFR gene mutations site T790M according to claim 1 a kind of, which is characterized in that
The extension primer and its variant is complementary with the sequence of mutational site upstream, 3 ' end last bit bases just with EGFR
C.2369C > base pair complementarity of the upstream T first, extension primer: 5 '-CGTGCAGCTCATCA-3 '.
6. a kind of inspection of the human EGFR gene mutations site T790M comprising the described in any item detection reagents of claim 1-5
Survey method, which is characterized in that this method can detect EGFR genetic mutation site T790M in blood or bodily tissue,
This method recycles extension primer specific amplification mutated gene by PCR amplification target fragment, and further magnetic particle technology is rich
Collection detects after improving accuracy and sensitivity.
7. the detection method of human EGFR gene mutations site T790M according to claim 6 a kind of, this method include with
Lower step:
1) sample preparation: paraffin-embedded tissue, flesh tissue freeze investing tissue, blood, and blood plasma and hydrothorax conventional treatment are extracted
DNA sample；
2) target fragment PCR amplification: PCR reaction solution I, PCR reaction solution II being added into reaction tube, and it is anti-to prepare PCR for DNA sample
Answer system；PCR reaction condition is arranged in PCR instrument, carries out PCR；
3) enzymic digestion: being added PCR reaction product and mix with enzymic digestion reaction solution, react under certain condition, after the reaction was completed, carries out
The inactivation of enzyme；
4) Single base extension: Single base extension liquid I being added into reaction tube, and Single base extension liquid II and PCR-SAP product carries out
PCR reaction condition is arranged in mixing, PCR instrument, carries out Single base extension；
5) enrichment with magnetic bead reacts: magnetic particle suspension is added in range by a certain percentage and extension products are mixed and are incubated for for the first time,
Product, which is placed on magnetic frame after magnetic bead adsorbs completely, discards supernatant liquid, and cleans magnetic bead with HPLC- water, and magnetic bead is resuspended in biology
It is incubated for for second in plain coating buffer, carries out desalination after rear of short duration centrifugation；
6) point sample is analyzed with data: after the sample point sample after desalination is mixed to CPM chip with matrix, carrying out Mass Spectrometer Method and data
Analysis.
8. the detection method of human EGFR gene mutations site T790M according to claim 7 a kind of, which is characterized in that
The temperature that second is incubated in the step 5) is 90 DEG C, incubation time 5min.
9. the detection method of human EGFR gene mutations site T790M according to claim 8 a kind of, which is characterized in that
It is that desalination is carried out by resin in the step 5).
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