CN109097470A - A kind of EGFR mutated gene detection method based on fluorescence-encoded micro-beads - Google Patents
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Abstract
The EGFR mutated gene detection method based on fluorescence-encoded micro-beads that the invention discloses a kind of, is related to the detection of EGFR mutated gene.The EGFR mutated gene detection method based on fluorescence-encoded micro-beads that the invention proposes a kind of, a variety of specificity detection probes for EGFR mutated gene are first separately fixed on different fluorescence-encoded micro-beads by this method, then the segment containing mutated gene in measuring samples is expanded by modified ARMS-PCR, the segment after amplification is hybridized with a variety of coding microballs that surface secures particular detection probe finally, and achievees the purpose that judge whether contain the specific mutational site EGFR in measuring samples by flow cytometry or Imaging-PAM.The invention also proposes a kind of for detecting the kit of EGFR mutated gene.The advantages of present invention has high sensitivity, accuracy height, high specificity, can detect a variety of EGFR mutated genes simultaneously.
Description
Technical field
The present invention relates to cancer patient's EGFR mutated gene detection fields, more particularly to one kind to be based on fluorescence-encoded micro-beads
EGFR mutated gene detection method.
Background technique
Human epidermal growth factor (epidermal growth factor receptor, EGFR) is a kind of albumen junket ammonia
Acid kinase receptor is distributed widely in the cell surfaces such as mammalian epithelial cell, fibroblast, spongiocyte, has junket ammonia
Kinase activity.EGFR is one of HER/ErbB family member.EGFR forms dimerization in cell surface with after its ligand binding
Body, by the activity of tyrosine kinase, activated receptor autophosphorylation, the cascade by adaptin and enzyme in cytoplasm is anti-
It answers, adjusts the transcription of transcription factor activation gene, instruct cell migration, increment, differentiation and apoptosis.When EGFR mutates,
EGFR itself or ligand are overexpressed, to stimulate cell to form runaway increment by autocrine or paracrine mode.In addition,
EGFR overactivity can also start the expression of multiple protein hydrolysis and angiogenic factors, to accelerate turning for cancer cell
It moves.Therefore, the usual prognosis of EGFR overexpression person is poor.
Iressa and Erlotinib are U.S. FDA approvals for NSCLC target as EGFR receptor tyrosine kinase inhibitors
To the key agents for the treatment of.But clinical trial shows that Iressa and Erlotinib only have significantly patient NSCLC of 10-30%
Curative effect.Further investigation revealed that EGFR genetic mutation is related to NSCLC targeted therapy curative effect, majority carries EGFR gene
The patient of mutation is significant in efficacy.There is the trouble of EGFR tyrosine kinase gene code area 18-21 exons mutation in lung carcinoma cell
The effective percentage of person, targeted drug Gefitinib are up to 80%.If patient does not carry EGFR genetic mutation and takes such medicine
Object not only can delay the state of an illness also and will cause the waste of huge medication resource.Therefore, it detects and whether contains in tissue or blood
EGFR genetic mutation has important reference value and future tumors individuation for instructing NSCLC patients clinical medication
The developing direction for the treatment of.
However EGFR sports somatic mutation, the frequency being mutated in tumor patient is higher (in U.S. NSCLC patient
About 10% has an EGFR genetic mutation, and 30%) asian population this ratio is about.But mutated gene with do not mutate
Wild type gene is compared, and mutant proportion is extremely low, and according to different neoplasm stagings, this ratio is usually 0.01%~10%.Therefore
The maximum problem of detection for EGFR mutated gene is how under a large amount of wild type gene backgrounds to detect minute quantity
The EGFR gene of mutation.In addition, more than 40 kinds can be up to the related EGFR mutation type of targeted therapy due to known, such as
What realizes that single effectively detects another challenge that 40 various mutations gene types are also EGFR detection technique.Detection base at present
Because the method for mutation specifically includes that direct sequencing, traditional fluorescence quantitative PCR method, high-resolution solubility curve method and probe
Amplification retardance mutation method (amplification refractory mutation system, ARMS).PCR sequencing PCR time-consuming and
Sensitivity is low, and the gene mutation for being usually more than 5% to content detects.Therefore this method is not suitable for being applied to clinical sample
Analysis.Traditional fluorescence quantitative PCR method is difficult to the expansion for making mutated genes obtain specificity in a large amount of wild type gene
Increase, is easy to appear the testing result of false positive；The flux of the bit number of points of detection and detection sample is all relatively small simultaneously.
High-resolution solubility curve method detection sensitivity is 5% or so, and used instrument and corresponding matched comparison are special
Very, while testing result stability is bad, it is difficult to be popularized in hospital.ARMS-PCR is 3 ' the end alkali using PCR primer
Base must the principle that could effectively expand complementary with template, by design be directed to mutational site specific PCR amplimer,
And then achieve the purpose that detect mutated gene.Although ARMS-PCR method is a kind of relatively simple and with higher sensitivity detection
Method, but it is constrained to PCR sense channel, it is difficult to it realizes and a variety of EGFR mutated genes is carried out while being detected.
The existing multiple determination system based on suspended array chip uses the microballoon containing encoded information, has
The advantage of qualitative and quantitative analysis can be carried out simultaneously to protein, nucleic acid and cell factor etc., therefore be widely used in life section
Learn research, disorder in screening, drug screening and clinical diagnosis.The core component of suspension array technology is with unique code letter
The microballoon of breath.These microballoons can pass through the optical materials such as organic fluorescent dye, Raman molecular, quantum dot, polymer quantum dot
Carry out optical encoding.
It is low with detection flux in order to solve the detection sensitivity difference that existing EGFR mutated gene detection technique is faced simultaneously
The problem of, the invention proposes a kind of to be directed to most common three kinds of mutation in EGFR mutated gene based on fluorescence-encoded micro-beads
The single tube multiple detection method of type (19Del, T790M and L858R cover 32 kinds of hypotypes).This method propose one kind to contain
There is the modified ARMS- of the ARMS-PCR primer sets and specificity blocker group for expanding measuring samples genetic fragment
PCR amplification system, and it is fixed with the multi-fluorescence-encoded micro-beads detection architecture of particular detection probe, and a kind of for detecting
The kit of EGFR mutated gene.The present invention has high sensitivity, accuracy height, high specificity, can detect simultaneously a variety of
The advantages of EGFR mutated gene.
Summary of the invention
In view of the above drawbacks of the prior art, it can be detected simultaneously technical problem to be solved by the invention is to provide a kind of
The detection method in a variety of mutational sites EGFR, this method can guarantee simultaneously the accuracy of sensitivity and the detection of detection method with
And detection flux, while this method is also easy to large-scale promotion.
To achieve the above object, the present invention provides a kind of EGFR mutated gene detection side based on fluorescence-encoded micro-beads
Method, which comprises the following steps:
Step 1: preparation is directed to the specificity detection probe in the different mutational sites EGFR, the detection probe includes 15-
The base sequence of 35bp, the medium position of the base sequence include the corresponding corresponding base sequence of EGFR mutated gene；
Step 2: by being separately fixed at for the detection probe in the different mutational sites EGFR with different coding information
On fluorescence-encoded micro-beads, the fluorescence-encoded micro-beads can be in conjunction with simultaneously fixed test probe；
Step 3: preparation corresponds to the amplimer in the different mutational sites EGFR and for different loci to improve reaction
The blocker of specificity, at least one primer in the pair of primer are marked using reporter fluorescence molecule, then sharp
With the segment containing mutated gene in modified ARMS-PCR system amplification measuring samples；
Step 4: the resulting fluorescence for being fixed with detection probe of genetic fragment and second step that third step amplification obtains is compiled
Code microballoon is hybridized, so that reaching fluorescence-encoded micro-beads can the specific purpose for capturing purpose amplified fragments；
5th step detects the encoded information of fluorescence-encoded micro-beads and micro- by flow cytometry or Imaging-PAM
The reporter fluorescent signal of ball surface determines that measuring samples whether there is and corresponding EGFR mutation type.
Preferably, the EGFR mutated gene detection method based on fluorescence-encoded micro-beads, which is characterized in that described
The different mutational sites EGFR include 19Del, at least one of T790M and L858R.
Preferably, the EGFR mutated gene detection method based on fluorescence-encoded micro-beads, which is characterized in that described
Fluorescent tag molecule is another reporter fluorescence molecule different from fluorescence-encoded micro-beads emission spectrum in third step.
Preferably, the marker can issue signal by way of fluorescence method, and eventually by flow cytometry or
Person's Imaging-PAM detects.
Preferably, the EGFR mutated gene detection method based on fluorescence-encoded micro-beads, which is characterized in that described
Detection probe is at least one sequence or its reverse complementary sequence in SEQ ID 1-ID 3.
Preferably, the EGFR mutated gene detection method based on fluorescence-encoded micro-beads, which is characterized in that described
Amplimer in third step is at least 1 couple: the SEQ ID 4 and SEQ ID 5 of following 3 centering；SEQ ID 6 and SEQ ID 7；
SEQ ID 8 and SEQ ID 9.SEQ ID 4 and the corresponding mutation type of SEQ ID 5 are 19Del；SEQ ID 6 and SEQ ID
7 corresponding mutation types are L858R；SEQ ID 8 and the corresponding mutation type of SEQ ID 9 are T790M.
Preferably, the EGFR mutated gene detection method based on fluorescence-encoded micro-beads, which is characterized in that described
It further include blocker sequence in third step modified ARMS-PCR amplification system, the sequence of the blocker contains above-mentioned
Gene mutation site, length 10bp-25bp modifies containing PNA or LNA in the blocker base.
Preferably, the EGFR mutated gene detection method based on fluorescence-encoded micro-beads, which is characterized in that described
Blocker sequence is at least one sequence or its reverse complementary sequence in 10-ID 12 of SEQ ID.Described in each
Blocker corresponds to the EGFR mutated gene type described at least one, wherein the corresponding mutation type of SEQ ID 10 is
The corresponding mutation type of EX19DEL, SEQ ID 11 is L858R, and the corresponding mutation type of SEQ ID 12 is T790M.
The present invention also provides a kind of kits for detecting EGFR mutated gene, it is characterised in that the detection EGFR is prominent
The kit for becoming gene is utilized above-mentioned any one method and is detected, and the kit includes being fixed with multiple specificity
The solid phase carrier of detection probe, the modified ARMS-PCR amplification system containing multiple amplimers and multiple blocker.
The present invention is compared with the prior art beneficial effect: the present invention is compiled based on modified ARMS-PCR and fluorescence
EGFR genetic mutation detection technique on code microballoon hybridisationdetection technology.Due to depositing for modified ARM-PCR and Blocker
The mutated gene type detected needed for it can be directed to carries out specific amplification, then by being fixed on fluorescence-encoded micro-beads
Detection probe to amplified production carry out hybridization check.Pass through the amplification strategy of modified ARMS-PCR combination Blocker
Joint fluorescence-encoded micro-beads hybridisationdetection technology can the gene mutation type directly to person to be checked can play good judgement, have
There are accuracy height, high specificity；Simultaneously also with the advantage of high detection sensitivity.
It is described further below with reference to technical effect of the attached drawing to design of the invention, specific structure and generation, with
It is fully understood from the purpose of the present invention, feature and effect.
Detailed description of the invention
In the fluorescence intensity of FL4 after the encoded microballon detection of Fig. 1 .L858R mutated gene sample；
Fig. 2 is the EGFR mutated gene detection method based on coding microball of a preferred embodiment of the invention
L858R sensitivity；
Fig. 3 is the more of the EGFR mutated gene detection method based on coding microball of a preferred embodiment of the invention
The detection sensitivity of weight modified-ARMS-PCR.
Specific embodiment
Technology contents of the invention are described further below with reference to embodiment: following embodiments be it is illustrative,
It is not restrictive, cannot be limited the scope of protection of the present invention with following embodiments.Test as used in the following examples
Method is conventional method unless otherwise specified.The materials, reagents and the like used in the following examples, unless otherwise specified,
Commercially obtain.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, such as Sambrook etc.
Molecular cloning: laboratory manual is shown in the versions in 1989 of New York:Cold Spring Harbor Laboratory Press
Described in condition, or according to the normal condition proposed by manufacturer.
EGFR mutated gene detection method of the embodiment 1 based on fluorescence-encoded micro-beads
Step 1: the preparation of the fluorescence-encoded micro-beads of coupling detection probe
1) the 100mM MES (PH 4.5 of 130-150 μ l is used；CAS Number:1266615-59-1) cleaning 0.25mg table
Face is fluorescence-encoded magnetic ball 3 times of carboxy functional group.
2) amido modified nucleic acid probe is added, then adds EDC (the CAS Number:25952- of fresh configuration
53-8) solution, so that EDC concentration is 40mg/ml.
3) it is protected from light 1h at room temperature, after reaction Magneto separate, supernatant is transferred to a new 0.5ml centrifuge tube
In.
4) it is cleaned magnetic ball 3 times with 130-150 μ l borate buffer solution (10mM, PH=9.5,0.5%SDS), then uses 130-
150 μ l 1xTE (PH7.6) are cleaned magnetic ball 3 times.
5) plus 125 μ l ddH2O are mixed (magnetic ball concentration is 2 μ g/ μ l), and 4 DEG C save.
Step 2: the amplification of measuring samples PCR
According to the positional relationship of this project EGFR mutated gene type to be checked, the present embodiment devises 3 pairs of primers and is directed to
Different mutated gene types is expanded simultaneously.The amplimer includes: SEQ ID 4 and SEQ ID 5；6 He of SEQ ID
SEQ ID 7；SEQ ID 8 and SEQ ID 9.SEQ ID 4 and the corresponding mutation type of SEQ ID 5 are 19Del；SEQ ID 6
It is L858R with the corresponding mutation type of SEQ ID 7；SEQ ID 8 and the corresponding mutation type of SEQ ID 9 are T790M.
5 ' ends of upstream primer and/or downstream primer are marked with fluorescein.Its pcr amplification product can be with present invention reality
Apply the probe hybridization in example.The present embodiment further includes blocker sequence, wherein each described blocker corresponding at least one
A EGFR mutated gene type, wherein the corresponding mutated gene type of SEQ ID 10 is EX19DEL, SEQ ID 11
Corresponding mutated gene type is L858R, and the corresponding mutated gene type of SEQ ID 12 is T790M.
Specific steps are as follows:
It extracts template DNA: extracting template DNA from the tissue samples of patient using other commercial kits.
Prepare PCR reaction solution: preparing the PCR reaction solution of 15 microlitres/person-portion, in the PCR reaction solution of every person-portion each ingredient and
Concentration be respectively as follows: upstream primer concentration be respectively 0.2 it is micro- rub/liter, Taq enzyme be 1U/ reaction, 1X PCR buffer, MgCl2For
1.5 mmoles/liter, dNTP be 0.2 mmoles/liter, template DNA 1-10 nanogram/microlitre and block sequence concentration 0.2 it is micro- rub/
It rises.
Modified-ARMS-PCR program are as follows: 95 DEG C initial denaturation 5 minutes；95 DEG C are denaturalized 30 seconds, 70 DEG C 20 seconds, 60 DEG C are multiple
Property 30 seconds, 72 DEG C extend 30 seconds, recycle 35 times.Product after PCR amplification includes the DNA fragmentation of patient to be detected.
Step 3: hybridization and Flow cytometry, specific steps are as follows:
1) PCR product is in 95 DEG C of denaturation 10min；In cooled on ice 5min.
2) hybridization system is configured, the coding microball that three kinds secure specific probe is added in hybridization buffer, so
The PCR product after denaturation is added afterwards, hybridizes 1h in 55 DEG C of Hybridization Ovens.
3) 2x SSC buffer solution for cleaning 1 time for using 130-150 μ l, is cleaned 1 time with 130-150 μ l 1x PBS liquid.
4) it is resuspended with the 1x PBS liquid of 35 μ l.
5) Flow cytometry analysis (setting detection 40 μ l of volume, shake before detection) Fig. 1 is fluidic cell point
Analyse result schematic diagram.
The detection sensitivity of EGFR mutated gene detection method of the embodiment 2 based on fluorescence-encoded micro-beads
By the wild template and various concentration (1,5.6,56,280 that are separately added into 10000 copies in reaction solution
With 560 copy) L858R mutated gene, then again by modified-ARMS PCR carry out augmentation detection with verify its spirit
Sensitivity (concrete outcome is detailed in Fig. 2).Testing result shows that it can reach 56 copies, and the wild base that can be copied to 10000
Because group can be carried out good inhibition.
In addition, passing through the saltant type of the wild template and different proportion that are separately added into 10000 copies in reaction solution
Gene (L858R, Del19 and T790M) then carries out augmentation detection by multiple modified-ARMS PCR again to verify it
Sensitivity (concrete outcome is detailed in Fig. 3).Testing result shows that it can reach 0.1%, and the wild base that can be copied to 10000
Because group can be carried out good inhibition.
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
Pass through the available skill of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Art scheme, all should be within the scope of protection determined by the claims.
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Claims (9)
1. a kind of EGFR mutated gene detection method based on fluorescence-encoded micro-beads, which comprises the following steps:
Step 1: preparation is directed to the specificity detection probe in the different mutational sites EGFR, the detection probe includes 15-35bp's
Base sequence, the medium position of the base sequence include the corresponding corresponding base sequence of EGFR mutated gene；
Step 2: being compiled the fluorescence with different coding information is separately fixed at for the detection probe in the different mutational sites EGFR
On code microballoon, the fluorescence-encoded micro-beads can be in conjunction with simultaneously fixed test probe；
Step 3: preparation corresponds to the amplimer in the different mutational sites EGFR and for different loci to improve atopic
Blocker, at least one primer in the pair of primer is marked using reporter fluorescence molecule, then utilized
Modified ARMS-PCR system expands the segment containing mutated gene in measuring samples；
Step 4: the genetic fragment that third step amplification is obtained with second step is resulting is fixed with the fluorescence-encoded micro- of detection probe
Ball is hybridized, so that reaching fluorescence-encoded micro-beads can the specific purpose for capturing purpose amplified fragments；
5th step detects the encoded information and microballoon table of fluorescence-encoded micro-beads by flow cytometry or Imaging-PAM
The reporter fluorescent signal in face determines that measuring samples whether there is and corresponding EGFR mutation type.
2. the EGFR mutated gene detection method based on fluorescence-encoded micro-beads as described in claim 1, which is characterized in that described
The different mutational sites EGFR include 19Del, at least one of T790M and L858R.
3. the EGFR mutated gene detection method based on fluorescence-encoded micro-beads as described in claim 1, which is characterized in that described
Fluorescent tag molecule is another reporter fluorescence molecule different from fluorescence-encoded micro-beads emission spectrum in third step.
4. the EGFR mutated gene detection method based on fluorescence-encoded micro-beads as claimed in claim 2, which is characterized in that described
Detection probe is at least one sequence or its reverse complementary sequence in SEQ ID 1-ID 3.
5. the EGFR mutated gene detection method based on fluorescence-encoded micro-beads as claimed in claim 2, which is characterized in that described
Amplimer in third step is at least 1 couple: the SEQ ID 4 and SEQ ID 5 of following 3 centering；SEQ ID 6 and SEQ ID 7；
SEQ ID 8 and SEQ ID 9.SEQ ID 4 and the corresponding mutation type of SEQ ID 5 are 19Del；SEQ ID 6 and SEQ ID 7
Corresponding mutation type is L858R；SEQ ID 8 and the corresponding mutation type of SEQ ID 9 are T790M.
6. the EGFR mutated gene detection method based on fluorescence-encoded micro-beads as claimed in claim 5, the PCR primer can be
Different loci is carried out while being expanded in single reaction pipe.
7. the EGFR mutated gene detection method based on fluorescence-encoded micro-beads as claimed in claim 2, which is characterized in that described
It further include blocker sequence in third step modified ARMS-PCR amplification system, the sequence of the blocker contains above-mentioned
Gene mutation site, length 10bp-25bp modifies containing PNA or LNA in the blocker base.
8. the EGFR mutated gene detection method based on fluorescence-encoded micro-beads as claimed in claim 7, which is characterized in that described
Blocker sequence is at least one sequence or its reverse complementary sequence in SEQ ID 10-SEQ ID 12.Described in each
Blocker corresponds to the EGFR mutated gene type described at least one, wherein the corresponding mutation type of SEQ ID 10 is
The corresponding mutation type of EX19Del, SEQ ID 11 is L858R, and the corresponding mutation type of SEQ ID 12 is T790M.
9. a kind of kit for detecting EGFR mutated gene, it is characterised in that the kit benefit of the detection EGFR mutated gene
With including the EGFR mutated gene detection method progress based on fluorescence-encoded micro-beads as described in claim 1-8 any one
Detection.
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| CN109811056A (en) * | 2019-02-28 | 2019-05-28 | 苏州唯善生物科技有限公司 | For colorectal cancer and its primed probe group and kit of precancerous lesion early diagnosis, detection or screening |
| CN111829940A (en) * | 2020-07-23 | 2020-10-27 | 四川大学 | Flow cytometry detection method for Pig-a gene mutation test in rat body |
| CN117106864A (en) * | 2023-07-11 | 2023-11-24 | 泛肽生物科技(浙江)有限公司 | Plasma free DNA methylation specific site and detection method of DMR section |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109811056A (en) * | 2019-02-28 | 2019-05-28 | 苏州唯善生物科技有限公司 | For colorectal cancer and its primed probe group and kit of precancerous lesion early diagnosis, detection or screening |
| CN111829940A (en) * | 2020-07-23 | 2020-10-27 | 四川大学 | Flow cytometry detection method for Pig-a gene mutation test in rat body |
| CN111829940B (en) * | 2020-07-23 | 2022-03-22 | 四川大学 | Flow cytometry detection method for rat peripheral blood Pig-a gene mutation test |
| CN117106864A (en) * | 2023-07-11 | 2023-11-24 | 泛肽生物科技(浙江)有限公司 | Plasma free DNA methylation specific site and detection method of DMR section |
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