CN109097469B - EGFR mutant gene detection method based on solid phase hybridization technology - Google Patents
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Abstract
The invention discloses an EGFR mutant gene detection method based on a solid phase hybridization technology, and relates to detection of EGFR mutant genes. The invention provides an EGFR mutant gene detection method based on a solid phase hybridization technology, which comprises the steps of fixing a plurality of specific detection probes aiming at EGFR mutant genes on a solid phase carrier, amplifying a fragment containing the mutant genes in a sample to be detected through modified ARMS-PCR, and hybridizing the amplified fragment with the probes on the solid phase carrier, thereby achieving the purpose of judging whether the sample contains specific EGFR mutant sites. The invention also provides a kit for diagnosing the EGFR mutant gene by using the method. The kit has the advantages of high sensitivity, high accuracy and strong specificity, and can simultaneously detect a plurality of EGFR mutant genes.
Description
Technical Field
The invention relates to the field of detection of EGFR mutant genes of cancer patients, in particular to an EGFR mutant gene detection method based on a solid phase hybridization technology.
Background
Human Epidermal Growth Factor (EGFR) is a protein tyrosine kinase receptor, widely distributed on the cell surfaces of epithelial cells, fibroblasts, glial cells and the like of mammals, and has tyrosine kinase activity. EGFR is one of the HER/ErbB family members. EGFR forms dipolymer on cell surface after being combined with ligand thereof, activates receptor autophosphorylation through the activity of tyrosine kinase, regulates the transcription of transcription factor activation gene through the cascade reaction of adaptor protein and enzyme in cytoplasm, and guides cell migration, proliferation, differentiation and apoptosis. When EGFR is mutated, EGFR itself or the ligand is overexpressed, thereby stimulating the cells to develop uncontrolled proliferation by autocrine or paracrine means. In addition, EGFR over-activation can also initiate the expression of various proteolytic and pro-angiogenic factors, thereby accelerating metastasis of cancer cells. Thus, EGFR overexpressors are often less prognostic.
Iressa and tarceva are major drugs approved by the FDA in the united states for the targeted treatment of NSCLC as EGFR receptor tyrosine kinase inhibitors. However, clinical experiments show that the Iressa and the Tarceva only have obvious curative effect on 10-30% of NSCLC patients. Further research finds that EGFR gene mutation is related to curative effect of NSCLC targeted therapy, and most patients carrying EGFR gene mutation have remarkable curative effect. The effective rate of the target medicine gefitinib reaches 80 percent for patients with EGFR tyrosine kinase gene coding region 18-21 exon mutation in lung cancer cells. If patients take the medicines without EGFR gene mutation, the illness state can be delayed, and a huge amount of medication resources can be wasted. Therefore, the detection of whether the tissue or the blood contains EGFR gene mutation has important reference value for guiding clinical medication of NSCLC patients, and is also a development direction of individualized treatment of tumors in the future.
EGFR mutations are somatic mutations, with a high frequency of mutations in tumor patients (approximately 10% of us NSCLC patients have EGFR gene mutations, compared to approximately 30% of asian populations). However, the mutant gene has a very low mutation ratio compared with the non-mutated wild-type gene, and the ratio is usually 0.01% to 10% depending on the stage of the tumor. Therefore, the biggest challenge in detecting mutant EGFR genes is how to detect very few mutant EGFR genes in a background of a large number of wild-type genes. In addition, since it is known that up to over 40 of the EGFR mutation types involved in treatment can be targeted, how to achieve effective detection of over 40 mutation types is another challenge. The current methods for detecting gene mutations mainly comprise: direct sequencing, conventional fluorescent quantitative PCR and probe Amplification Retardation Mutation (ARMS) (application No. CN101608240B/CN 102747157A). The direct sequencing method is long in time consumption and low in sensitivity, can only detect gene mutation with the content of more than 5 percent, can only be used for scientific research in laboratories generally, and is difficult to be used for clinical detection. The traditional fluorescent quantitative PCR method is difficult to ensure that the mutant gene is subjected to specific amplification in a large amount of wild genes, and a false positive detection result is easy to appear; the number of the detected sites and the flux of the detected samples are relatively small. The ARMS-PCR method is based on the principle that the 3' end base of the PCR primer must be completely complementary with the template for effective amplification, and the purpose of detecting the mutant gene is achieved by designing the specific PCR amplification primer aiming at the mutant site. Although the ARMS-PCR method is a simpler detection method with higher sensitivity, the ARMS-PCR method is limited by a PCR detection channel, and the simultaneous detection of a plurality of EGFR mutant genes is difficult to realize.
Therefore, in order to simultaneously solve the problems of low sensitivity and low detection flux of the existing EGFR mutant gene detection technology, the invention aims to develop a novel EGFR mutant site detection method and a kit. The method has the advantages of high sensitivity, high accuracy and strong specificity, and can simultaneously detect a plurality of mutant genes.
Disclosure of Invention
In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a detection method capable of simultaneously detecting multiple EGFR mutation sites; the method can simultaneously ensure the sensitivity, the detection accuracy and the detection flux of the detection method, and is easy for large-scale popularization.
The invention provides an EGFR mutant gene detection method based on a solid phase hybridization technology, which is characterized by comprising the following steps:
the first step is as follows: preparing specific detection probe sets aiming at different EGFR mutation sites, wherein the detection probe sets comprise 15-35bp base sequences, and the middle positions of the base sequences comprise corresponding base sequences of corresponding EGFR mutation genes;
the second step is that: respectively fixing detection probes aiming at different EGFR mutation sites on different positions of a solid phase carrier or fixing the detection probes on the solid phase carrier containing different coding information, wherein the solid phase carrier is required to be capable of combining and fixing the detection probes;
the third step: preparing amplification primers corresponding to different EGFR mutation sites and a blocker for improving reaction specificity aiming at different mutation sites, marking at least one primer in the pair of primers by using a marker, and amplifying a fragment containing a mutant gene in a sample to be detected by using a modified ARMS-PCR system;
the fourth step: hybridizing the gene fragment obtained by the amplification in the third step with the solid phase carrier which is obtained by the second step and is fixed with the detection probes with different specificities, thereby achieving the purpose of capturing the target amplification fragment;
and fifthly, determining whether the sample to be detected exists and the corresponding EGFR mutant gene type by adopting enzymatic color development, chemiluminescence or fluorescence according to the marker on the amplification primer.
The middle position refers to the middle when the number of the probe bases is odd, and refers to any one of the two in the middle when the number of the probes is even.
Preferably, the different EGFR mutation sites according to the present invention include at least one of G719D/G719A/G719S/G719C, 19DEL, EX20insert, T790M, S768I, L858R and L861Q.
Preferably, the specific detection probe has an amino group or biotin at the 3 'end or 5' end for immobilization with a solid support by chemical covalent linkage or biotin-avidin affinity.
Preferably, the solid phase carrier material can be at least one of glass, silica gel, ceramic, plastic, metal, solid phase porous membrane, nylon membrane, PVDF membrane or microspheres with different encoded information, but is not limited to the listed solid phase carriers. The solid support may be bound directly or indirectly to the detection probe. The solid-phase porous membrane, the nylon membrane or the PVDF membrane can be directly combined with the detection probe, and the surfaces of glass, silica gel, ceramics, plastics, metal or microspheres with different coded information and the like need to be processed to generate active functional groups which can be covalently combined with the detection probe.
Wherein the label in the third step can be detected by enzymatic color development, chemiluminescence or fluorescence, preferably, the label is biotin or fluorescein labeled and is labeled at the 5' end of the primer. The biotin can be combined with HRP (horse radish peroxidase) marked with avidin/streptavidin, and the detection of the marked primer is realized by a chemiluminescence method or a fluorescence method.
Preferably, the marker is marked at the 5' end of the primer, the marker is a fluorescein reporter molecule, the EGFR target mutant gene segment obtained by the third step of modified ARMS-PCR amplification is hybridized with the coding microspheres fixed with the specific detection probes, and the detection of the EGFR mutant gene is realized by detecting the coding information of the coding microspheres and the fluorescence intensity of the reporter fluorescent molecule by adopting a flow cytometry or fluorescence imaging method.
The detection probe is at least one sequence of SEQ ID 1-SEQ ID7 or a reverse complementary DNA sequence thereof, the mutation type corresponding to SEQ ID 1 is 19Del, the mutation type corresponding to SEQ ID 2 is G719D/G719A/G719S/G719C, the mutation type corresponding to SEQ ID3 is EX20insert, the mutation type corresponding to SEQ ID 4 is L858R, the mutation type corresponding to SEQ ID 5 is T790M, the mutation type corresponding to SEQ ID 6 is S768I, and the mutation type corresponding to SEQ ID7 is L861Q.
Preferably, the amplification primers in the third step are at least one of the following 17 pairs: SEQ ID 8 and SEQ ID 9; SEQ ID 10 and SEQ ID 11; SEQ ID 10 and SEQ ID 12; SEQ ID 10 and SEQ ID 13; SEQ ID 10 and SEQ ID 14; SEQ ID 15 and SEQ ID 16; SEQ ID 15 and SEQ ID 17; SEQ ID 15 and SEQ ID 18; SEQ ID 15 and SEQ ID 19; SEQ ID 15 and SEQ ID 20; SEQ ID 15 and SEQ ID 21; SEQ ID 22 and SEQ ID 23; SEQ ID 24 and SEQ ID 25; SEQ ID 26 and SEQ ID 27; SEQ ID 28 and SEQ ID 29.
Preferably, the amplification primers of the modified ARMS-PCR system can be combined in different ways to achieve simultaneous amplification of different mutation sites in a single reaction tube: SEQ ID 8/SEQ ID 9/SEQ ID 15/SEQ ID 16/SEQ ID 17/SEQ ID 18/SEQ ID 19/SEQ ID 20/SEQ ID 21 are capable of amplifying the type of mutation comprising 19Del and EX20insert simultaneously; SEQ ID 22/SEQ ID 23/SEQ ID 24/SEQ ID 25 are capable of amplifying mutation types comprising L858R and T790M simultaneously; SEQ ID 10/SEQ ID 11/SEQ ID 12/SEQ ID 13/SEQ ID 14/SEQ ID 26/SEQ ID 27/SEQ ID 28/SEQ ID 29 are capable of simultaneously amplifying the type of mutation comprising G719D/G719A/G719S/G719C/S768I/L861Q;
in a preferred embodiment of the invention, the third modified ARMS-PCR amplification system further comprises a blocker sequence of a mutation site to be detected, the length of the blocker sequence is 10bp-25bp, and the base group of the blocker sequence contains PNA or LNA modification. Preferably, the length of the blocker sequence is 10-15 bp.
The sequence of the blocker contains the gene mutation site, and the EGFR mutation site is at least one of 19DEL, G719D/G719A/G719S/G719C, T790M, S768I, L858R and L861Q; the block sequence is at least one of the 6 blocks. The blocker base contains PNA or LNA modification.
Preferably, the blocker is at least one sequence of SEQ ID30-SEQ ID 35 or a reverse complementary sequence thereof.
Preferably, each of the blockers corresponds to at least one EGFR mutation gene type, wherein the mutation type corresponding to SEQ ID30 is 19Del, the mutation type corresponding to SEQ ID 31 is G719D/G719A/G719S/G719C, the mutation type corresponding to SEQ ID 32 is L858R, the mutation type corresponding to SEQ ID 33 is T790M, the mutation type corresponding to SEQ ID 34 is S768I, and the mutation type corresponding to SEQ ID 35 is L861Q.
Preferably, the sequence of the blocker is at least one sequence of SEQ ID 30/SEQ ID 31/SEQ ID 32/SEQ ID 33/SEQ ID 34/SEQ ID 35 or at least one sequence of a reverse complementary sequence thereof, each of the blockers corresponds to at least one of the EGFR mutant gene types, wherein the mutant gene type corresponding to SEQ ID30 is 19DEL, the mutant gene type corresponding to SEQ ID 31 is G719D/G719A/G719S/G719 38 719C, the mutant gene type corresponding to SEQ ID 32 is L858R, the mutant gene type corresponding to SEQ ID 33 is T790M, the mutant gene type corresponding to SEQ ID 34 is S768I, and the mutant gene type corresponding to SEQ ID 35 is L861Q. The blocker sequence can further inhibit the amplification of a wild type gene so as to improve the detection specificity of the method.
The invention also provides a kit for detecting EGFR mutation, which is characterized in that the kit for detecting EGFR mutation genes is used for detecting by any one of the methods, and the kit comprises a solid phase carrier fixed with a plurality of specific detection probes, and a modified ARMS-PCR amplification system containing a plurality of amplification primers and a plurality of blocks.
Compared with the prior art, the invention has the advantages that: the invention relates to an EGFR gene mutation detection technology based on modified ARMS-PCR and solid phase hybridization detection technology. Due to the existence of modified ARMS-PCR and Blocker, the mutation types to be detected can be specifically amplified, and then the amplified products can be subjected to hybridization detection by a detection probe fixed on the surface of a solid phase matrix. Therefore, the amplification strategy combining modified ARMS-PCR with Blocker and the solid-phase hybridization detection technology can directly and well judge the type of the mutant gene of a person to be detected, and the method has the advantages of high accuracy, strong specificity and high detection sensitivity.
Drawings
FIG. 1 shows the results of the detection of G719A on different wild type templates (10000 copies) and mutation ratios;
FIG. 2 shows the detection results of different mutant genome contents of the L858R detection reagent in 10000 wild-type background.
Detailed Description
The technical content of the invention is further explained by the following embodiments: the following examples are illustrative and not intended to be limiting, and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: a Laboratory manual is described in New York, Cold Spring Harbor Laboratory Press, 1989 edition, or as recommended by the manufacturer.
Example 1 EGFR mutation detection based on hybrid membrane strips (chips)
The first step is as follows: preparation of nucleic acid hybrid membrane strip (chip)
1. The array of sites is printed on a substrate (e.g., nylon membrane) with a printer and the probe names of the corresponding sites are indicated (see table 1 for probe details):
2. the membrane was soaked with a 5% EDAC (Sigma, CAS Number:25952-53-8) solution for 30 minutes to activate the carboxyl groups on the surface of the nylon membrane;
3. each of 7 probes was diluted to 5 μm/l with probe diluent (0.1 × TE);
4. according to a locus array printed on the nylon membrane, 7 probes are respectively and correspondingly spotted on corresponding loci of the nylon membrane according to the names of the probes, and amino groups on the probes can generate a cross-linking reaction with carboxyl on the surface of the nylon membrane;
5. after the nylon membrane is dried, soaking the membrane for 5 minutes by using 0.1 mol/L NaOH to seal unreacted carboxyl on the surface of the nylon membrane;
6. washing the nylon membrane with pure water, and drying for later use.
Table 1: probe sequences for mutation detection
The second step: multiplex modified ARMS-PCR amplification
According to the positional relationship of the EGFR mutant gene types to be detected in the project, corresponding primers are designed for different mutant gene types for amplification, and the details of the primers are shown in Table 2. Wherein SEQ ID 8/SEQ ID 9/SEQ ID 15/SEQ ID 16/SEQ ID 17/SEQ ID 18/SEQ ID 19/SEQ ID 20/SEQ ID 21 are capable of amplifying a mutation type comprising 19DEL and EX20insert simultaneously; SEQ ID 22/SEQ ID 23/SEQ ID 24/SEQ ID 25 are capable of amplifying mutation types comprising L858R and T790M simultaneously; SEQ ID 10/SEQ ID 11/SEQ ID 12/SEQ ID 13/SEQ ID 14/SEQ ID 26/SEQ ID 27/SEQ ID 28/SEQ ID 29 are capable of simultaneously amplifying the type of mutation comprising G719D/G719A/G719S/G719C/S768I/L861Q;
table 2: primer sequences required for ARMS-PCR amplification
The 5' end of the upstream primer and/or the downstream primer is provided with biotin or a report fluorescent label. The embodiment of the invention is to carry out biotin labeling on the upstream primer, and the PCR amplification product can be hybridized with the nucleic acid hybridization membrane strip fixed with the detection probe.
Extracting template DNA: other commercial kits are used to extract template DNA from a tissue sample from a patient.
Synthesizing a PCR primer: the synthesis method is the conventional DNA synthesis method, and biotin labels are added at the 5' end of the upstream primer.
Preparing modified ARMS-PCR reaction solution: preparing 15 microliter/person parts of PCR reaction solution, wherein the components and the concentrations in each person part of PCR reaction solution are as follows: the concentrations of the upstream and downstream primers are respectively 0.2 micromole/liter, 1U of Taq enzyme per reaction, 1 XPCR buffer and MgCl21.5 mmol/l, dNTP 0.2 mmol/l, template DNA 1-10 ng/l, and the concentration of the block sequence is 0.2. mu.m/l, and the block sequence is shown in Table 3.
Table 3: block sequence required in ARMS-PCR amplification
The modified ARMS-PCR amplification program is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 sec, renaturation at 70 ℃ for 20 sec, renaturation at 60 ℃ for 30 sec, extension at 72 ℃ for 30 sec, and 35 cycles. The PCR amplified product contains the DNA fragment of the patient to be tested.
The third step: solid phase hybridization and enzymatic chromogenic assay
1. Hybridization of
The product of modified ARMS-PCR amplification and the hybridization membrane strip immobilized with the detection probe were added to 1ml of a hybridization solution (2 XSSC, 0.1% SDS, pH7.4) and hybridized at 42 ℃ for 30 minutes in a hybridization chamber.
2. Washing membrane
The hybridized membrane strips were transferred to a wash (0.5 XSSC, 0.1% SDS, pH7.4) preheated to 42 ℃ and washed at 42 ℃ for 15 minutes.
3. Peroxidase (HRP) incubation
Putting the hybrid membrane strip into a freshly prepared 0.25U/ml avidin-HRP solution, and incubating for 15 minutes at 37 ℃ to combine avidin with biotin in the PCR product, so that the HRP can be connected to the PCR product; the excess avidin-HRP is washed away by a washing process.
4. Color development
Preparing color developing solution, placing the film strip into color developing solution (0.1 mol/L sodium citrate, 0.1 mg/mL TMB, 0.0015% H)2O2) The membrane strip is protected from light and develops color for 15 minutes, and blue spots appear at corresponding probe positions with hybridization products on the membrane strip.
5. Result judgment
The color development result of the membrane strip can be judged by naked eyes, and whether the gene mutation exists in the sample to be detected is judged according to the existence of color development signals on different probe positions on the hybridized membrane strip. If a blue spot appears, the mutation type can be judged to be positive, i.e., the mutation type exists. In this example, the detection sensitivity of G719A can reach 1% (in 10000 copies of wild type template background) (FIG. 1, wherein: 1.1% mutant template; 2.200 copies of mutant template; 3. wild 10000; 4. negative control). The detection sensitivity of the modified ARMS-PCR combined nucleic acid hybrid membrane strip for different mutation sites of EGFR is about 1%, and the specific information is shown in Table 4.
TABLE 4 sensitivity of EGFR mutant gene detection method based on solid phase hybridization technique for detecting different mutant sites
| Mutation | Detection line | ||
| 1 | L858R | 0.20％ | |
| 2 | T790M | 1.0％ | |
| 3 | S768I | 1.0％ | |
| 4 | G719S | Less than 1.0% | |
| 5 | G719A | 1.0％ | |
| 6 | G719D | Less than 1.0 percent | |
| 7 | G719C | 1.0％ | |
| 8 | L861Q | 1.0％ | |
| 9 | 19Del | 1.0％ | |
| 10 | 20insert | 1.0％ |
Example 2 EGFR mutant Gene detection based on fluorescent-encoded microspheres
The first step is as follows: preparation of fluorescent coding microsphere of coupling detection probe
1) Using 130-; CAS Number 1266615-59-1) Wash 0.25mg fluorescent-coded magnetic spheres 3 times.
2) The amino-modified nucleic acid probe was added, followed by a freshly prepared solution of EDC (CAS Number:25952-53-8) such that the EDC concentration was 40 mg/ml.
3) And (4) carrying out the reaction for 1h in the absence of light at room temperature, carrying out magnetic separation after the reaction is finished, and transferring the supernatant into a new 0.5ml centrifuge tube.
5) The beads were washed 3 times with 130-150. mu.l borax buffer (10mM, pH 9.5, 0.5% SDS) and 3 times with 130-150. mu.l 1XTE (pH 7.6).
6) Add 125. mu.l ddH2O and mix well (magnetic sphere concentration 2. mu.g/. mu.l), and store at 4 ℃.
The second step is that: PCR amplification of samples to be tested
According to the positional relationship of the EGFR mutant gene types to be detected in the project, 2 pairs of primers are designed in the embodiment and can simultaneously amplify different mutant gene types, and the SEQ ID 22/SEQ ID 23/SEQ ID 24/SEQ ID 25 can simultaneously amplify the mutant types containing L858R and T790M.
The 5' end of the upstream primer is provided with a fluorescein label. The PCR amplification product can be hybridized with the probe in the embodiment of the invention. The embodiment also comprises a corresponding blocker sequence, SEQ ID 32 and SEQ ID 33, wherein the mutant gene type corresponding to the SEQ ID 32 is L858R, and the mutant gene type corresponding to the SEQ ID 33 is T790M.
The specific operation steps are as follows:
extracting template DNA: other commercial kits are used to extract template DNA from patient tissue samples.
Preparing a PCR reaction solution: preparing 15 microliter/person PCR reaction solution, wherein the components and the concentrations in each person PCR reaction solution are respectively as follows: the concentrations of the upstream primer and the Taq enzyme are respectively 0.2 micromole/liter, 1U/reaction, 1X PCR buffer and MgCl21.5 mmol/l, 0.2 mmol/l dNTP, 1-10 nm template DNAG/l and a block sequence concentration of 0.2. mu.M.
The modified ARMS-PCR amplification program is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 seconds, renaturation at 70 ℃ for 20 seconds, renaturation at 60 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and 35 cycles. The PCR amplified product contains the DNA fragment of the patient to be detected.
The third step: the hybridization and flow cytometry detection comprises the following specific operation steps:
1) the PCR product was denatured at 95 ℃ for 10 min; cool on ice for 5 min.
2) Preparing a hybridization system, adding two coded microspheres fixed with specific probes into a hybridization buffer solution, then adding a denatured modified ARMS-PCR product, and hybridizing for 1h in a molecular hybridization instrument at 55 ℃.
3) Wash 1 time with 130-150 μ l 2 XSSC buffer and 1 time with 130-150 μ l 1 XSPBS.
4) Resuspend with 35. mu.l of 1 XPBS.
5) Flow cytometry analysis (setting detection volume 40. mu.l, shaking before detection)
FIG. 2 is the detection result of the L858R detection reagent on the content of different mutant genomes in 10000 wild-type background, wherein: (1) all wild plants; (2) presence of 0.1% mutant; (3) presence of 1% mutant; (4) there was 10% mutation.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
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<213> Artificial Sequence (Artificial Sequence)
<400> 14
gtgccgaacg caccggagg 19
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
ttctggccac catgcgaagc 20
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gggttatcca cgctggccac 20
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gttgtccacg ctgtccacgc 20
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gcacacgtgg gggttaccgt 20
<210> 19
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gcggcacacg tggtggggg 19
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ggttgtccac gctggccacg 20
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ggttgtccac gctggttacg 20
<210> 22
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
gctgacctaa agccacctcc ttac 24
<210> 23
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
tgtcaagatc acagattttg ggcg 24
<210> 24
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ctccaccgtg carctcatca t 21
<210> 25
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ttgagcaggt actgggagcc aat 23
<210> 26
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
ctccaggaag cctacgtgat ggccat 26
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gtggaggtga ggcagatgcc 20
<210> 28
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
cttggtgcac cgcgacctg 19
<210> 29
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
tctttctctt ccgcacccag ct 22
<210> 30
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
aattaagaga agcaacat 18
<210> 31
<211> 11
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ggagcccagc a 11
<210> 32
<211> 13
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
ttgggctggc caa 13
<210> 33
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
ctcatcacgc agctc 15
<210> 34
<211> 12
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
tggccagcgt gg 12
<210> 35
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
cacccagcag tttgg 15
Claims (9)
1. A kit for detecting EGFR mutant gene is characterized in that the kit for detecting EGFR mutant gene utilizes an EGFR mutant gene detection method based on solid phase hybridization technology to detect; the EGFR mutant gene detection method based on the solid phase hybridization technology comprises the following steps:
the first step is as follows: preparing specific detection probes aiming at different EGFR mutation sites, wherein the detection probes comprise 15-35bp base sequences, and the middle positions of the base sequences comprise corresponding base sequences of corresponding EGFR mutation genes;
the second step is that: respectively fixing detection probes aiming at different EGFR mutation sites on different positions of a solid phase carrier or fixing the detection probes on the solid phase carrier containing different coding information, wherein the solid phase carrier is required to be capable of combining and fixing the detection probes;
the third step: preparing amplification primer groups corresponding to different EGFR mutation sites and a blocker group aiming at different mutation sites and used for improving reaction specificity, wherein at least one primer in the pair of primers is marked by a marker, and then amplifying a fragment containing a mutant gene in a sample to be detected by utilizing a modified ARMS-PCR system;
the fourth step: hybridizing the gene fragment obtained by the amplification in the third step with the solid phase carrier which is obtained by the second step and is fixed with the detection probes with different specificities, thereby achieving the purpose of capturing the target amplification fragment;
fifthly, determining whether the sample to be detected exists and the corresponding EGFR mutant gene type by adopting enzymatic color development, chemiluminescence or fluorescence according to the marker on the amplification primer;
the kit comprises the solid phase carrier fixed with a plurality of the specificity detection probes, and the modified ARMS-PCR amplification system containing a plurality of the amplification primers and a plurality of the blockers.
2. The kit of claim 1, wherein the different EGFR mutation sites comprise at least one of G719D, G719A, G719S, G719C, 19-del, EX20insert, T790M, S768I, L858R, and L861Q.
3. The kit of claim 1, wherein the solid support is at least one of glass, silica gel, ceramic, plastic, metal, solid porous membrane, nylon membrane, PVDF membrane, or microspheres with different encoded information, and the solid support can be directly or indirectly bound to the specific detection probe.
4. The kit of claim 1, wherein the label in the third step is detectable by enzymatic color development, chemiluminescence, or fluorescence.
5. The kit of claim 2, wherein the detection probe is at least one sequence of SEQ ID 1-SEQ ID7 or a reverse complement thereof, the mutation type corresponding to SEQ ID 1 is 19DEL, the mutation type corresponding to SEQ ID 2 is G719D/G719A/G719S/G719 38 719C, the mutation type corresponding to SEQ ID3 is EX20insert, the mutation type corresponding to SEQ ID 4 is L858R, the mutation type corresponding to SEQ ID 5 is T790M, the mutation type corresponding to SEQ ID 6 is S768I, and the mutation type corresponding to SEQ ID7 is L861Q.
6. The kit of claim 2, wherein the amplification primers in the third step are at least one of the following 17 pairs: SEQ ID 8 and SEQ ID 9; SEQ ID 10 and SEQ ID 11; SEQ ID 10 and SEQ ID 12; SEQ ID 10 and SEQ ID 13; SEQ ID 10 and SEQ ID 14; SEQ ID 15 and SEQ ID 16; SEQ ID 15 and SEQ ID 17; SEQ ID 15 and SEQ ID 18; SEQ ID 15 and SEQ ID 19; SEQ ID 15 and SEQ ID 20; SEQ ID 15 and SEQ ID 21; SEQ ID 22 and SEQ ID 23; SEQ ID 24 and SEQ ID 25; SEQ ID 26 and SEQ ID 27; SEQ ID 28 and SEQ ID 29.
7. The kit of claim 6, wherein the PCR amplification primers can be combined in different ways to simultaneously amplify different sites in a single reaction tube: SEQ ID 8/SEQ ID 9/SEQ ID 15/SEQ ID 16/SEQ ID 17/SEQ ID 18/SEQ ID 19/SEQ ID 20/SEQ ID 21 are capable of amplifying mutation types comprising 19DEL and EX20insert simultaneously; SEQ ID 22/SEQ ID 23/SEQ ID 24/SEQ ID 25 are capable of amplifying mutation types comprising L858R and T790M simultaneously; SEQ ID 10/SEQ ID 11/SEQ ID 12/SEQ ID 13/SEQ ID 14/SEQ ID 26/SEQ ID 27/SEQ ID 28/SEQ ID 29 enable the simultaneous amplification of the type of mutation comprising G719D/G719A/G719S/G719C/S768I/L861Q.
8. The kit of claim 2, wherein the third modified ARMS-PCR amplification system further comprises a corresponding blocker sequence, the blocker sequence contains the gene mutation site, the length is 10bp to 25bp, and the base of the blocker sequence contains PNA or LNA modification.
9. The kit of claim 8, wherein the sequence of the blocker is at least one of SEQ ID30-SEQ ID 35 or a reverse complementary DNA sequence thereof, each of the blockers corresponds to at least one of the EGFR mutant gene types, wherein the mutation type corresponding to SEQ ID30 is 19DEL, the mutation type corresponding to SEQ ID 31 is G719D/G719A/G719S/G719C, the mutation type corresponding to SEQ ID 32 is L86858 25, the mutation type corresponding to SEQ ID 33 is T790M, the mutation type corresponding to SEQ ID 34 is S768I, and the mutation type corresponding to SEQ ID 35 is L861Q.
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| CN112522406B (en) * | 2020-12-10 | 2024-03-01 | 珠海澳加动力生物科技有限公司 | EGFR gene mutation detection system |
| CN114592045B (en) * | 2022-04-29 | 2024-03-26 | 江苏吉诺思美精准医学科技有限公司 | Primer and probe composition for detecting EGFR gene mutation by multiple fluorescence pcr, kit and detection method |
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| WO2013190089A1 (en) * | 2012-06-21 | 2013-12-27 | Pangaea Biotech, S.L. | Molecular biomarkers for predicting outcome in lung cancer |
| CN108251511A (en) * | 2018-03-31 | 2018-07-06 | 邓定平 | The detection kit and detection method of a kind of EGFR genetic mutation |
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Application publication date: 20181228 Assignee: Joinstar Biomedical Technology Co.,Ltd. Assignor: JIANGSU NANOFE BIOMEDICAL TECH. Co.,Ltd. Contract record no.: X2020980004307 Denomination of invention: EGFR (epidermal growth factor receptor) mutant gene detection method based on solid phase hybridization technology License type: Exclusive License Record date: 20200723 |
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