CN106755297A - One group is based on primer sets that ARMS fluorescence quantitative PCR detection EGFR genes T790M is mutated and preparation method thereof - Google Patents
One group is based on primer sets that ARMS fluorescence quantitative PCR detection EGFR genes T790M is mutated and preparation method thereof Download PDFInfo
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Abstract
The one group disclosed by the invention primer sets for being used to detect EGFR gene T790M point mutation, it includes following primer：T790M‑F:CTCCACCGTGCAGCTCATCTT；T790M‑R:TGCACACACCAGTTGAGCAGGT；TaqMan MGB probes 1：TCGGCTGCCTCCTGGA；Wherein T790M R are wild type anti-sense primer, and sense primer T790M F are ARMS primers, and base mismatch T is additionally introduced in 3 ' end seconds, are made for wild-type template, there is 2 base mismatch, and amplification efficiency is decreased obviously；And for the template that T790M is mutated, only one of which mispairing, and not in 3 ' ends, remain to specific amplification；So as in Sensitive Detection 5ng samples as little as 1% T790M mutation.The invention also discloses above-mentioned primer and the preparation method of probe.Beneficial effects of the present invention：1. high specific；2. quick, efficient amplification；3. sensitivity is high；4. identify easy；5. purposes is wide.
Description
Technical field
The invention belongs to molecule diagnosis and gene diagnosis field, it is related to a kind of for detecting Patients with Non-small-cell Lung sample
One group of middle EGFR gene T790M point mutation be based on ARMS fluorescence quantitative PCR detection EGFR genes T790M mutation primer sets and
Its preparation method.
Background technology
Non-small cell lung cancer (non-small cell lung cancer, NSCLC) is the normal of serious harm human health
See malignant tumour.In recent years, epidermal growth factor recipient tyrosine kinase inhibitor (epidermal growth
Factorreceptor-tyrosine kinase inhibitor, EGFR-TKI) obtain wide in the treatment of NSCLC patient
General application.
EGFR-TKI can effectively delay the progress of advanced NSCLC and extend patient survival.The curative effect of EGFR-TKI is very
The mutation status of EGFR are depended in big degree.Early clinic research display, women, asian ancestry, non-smoking or few smoking, gland cancer
NSCLC patient has reactivity higher to Gefitinib (Gefitinib).It is subsequently found, EGFR is mutated the treatment with Gefitinib
Effect has obvious correlation.Mutation on 18/19/20/21 extron and EGFR- in Patients with Non-small-cell Lung EGFR gene
The therapeutic effect of TKI is significantly correlated.Wherein 18, sport drug susceptibility mutation, 20 exons on 19,21 exons
On sport medicament-resistant mutation.
The issue of National Cancer integrated network《Non-small cell lung cancer NCCN guides》In point out, it is sensitive prominent for EGFR
The patient of change a, line recommends Gefitinib, Tarceva and Afatinib, and compared with chemotherapy, targeted therapy has more preferably
Curative effect and less toxic and side effect.But after targeted drug takes about 8-16 month, patient can typically occur resistance.It is wherein main
It is EGFR T790M medicament-resistant mutations so that Patients with Non-small-cell Lung produces resistance to Gefitinib and Tarceva, but
Osimertinib (AZD9291) has good therapeutic effect to the patient for carrying EGFR T790M.Therefore the point to T790M is dashed forward
Become detection, contribute to direction of medication usage.
At present, EGFR mutation detection method still with tissue examination as goldstandard, but tissue specimen mutation detection due to
The tissue mass of acquisition is not enough, and it is undesirable to obtain tissue site, therefore with certain limitation.In addition with sanger PCR sequencing PCRs,
The methods such as PCR-RFLP methods, PCR-SSCP methods, ARMS methods, Real-time PCR methods, DHPLC.Wherein, sanger PCR sequencing PCRs can
Directly to obtain sample abrupt information by sequencing, but the method requirement sample is tumor tissues, and sensitiveness is not high, can only
Mutation of the frequency of mutation more than 10% is detected, is easily influenceed by background dna, should not be used in other outside tumor tissues
Sample.In addition to direct sequencing, PCR-SSCP and PCR-RFLP methods also only see the detection for tumor tissues, there are no
The introduction detected in other samples.ARMS fluorescence quantitative PCR methods have high specificity, sensitivity high, reproducible, can be with standard
It is 1% even lower mutation really to detect the frequency of mutation, and the used time is short, and only needing 2 hours or so can be with the detection of complete paired samples.
Therefore present invention utilizes the above-mentioned advantage of the technology, it is applied to the detection of EGFR gene T790M point mutation.
By 3 ' end designs in mutational site during ARMS design of primers, last base is matched with mutating alkali yl, only the end of primer 3 '
When matching completely, could normally expand, when the end of primer 3 ' occurs mispairing, it is impossible to effectively amplification.But for Substitution,
T790M such as EGFR is mutated, and the ARMS primers matched completely with mutagenesis template still can occur effective with wild type DNA as template
Amplification.
The content of the invention
The technical problems to be solved by the invention are directed to existing for the method for detection EGFR gene T790M mutation at present
It is not enough and a kind of primer sets based on ARMS fluorescence quantitative PCR detection EGFR genes T790M mutation are provided.
The technical problems to be solved by the invention can be achieved through the following technical solutions：
One group of primer sets for being used to detect EGFR gene T790M point mutation, it includes following primer：
T790M-F:CTCCACCGTGCAGCTCATCTT；
T790M-R:TGCACACACCAGTTGAGCAGGT；
TaqMan-MGB probes 1：TCGGCTGCCTCCTGGA；
Wherein T790M-R is wild type anti-sense primer, and sense primer T790M-F is ARMS primers, and second volume is held 3 '
Outer introducing base mismatch T, makes for wild-type template, there is 2 base mismatch, and amplification efficiency is decreased obviously；And it is prominent for T790M
The template of change, only one of which mispairing, and do not remain to specific amplification in 3 ' ends；So as to Sensitive Detection 5ng DNA samples
In as little as 1% T790M mutation.
In a preferred embodiment of the invention, EGFR without mutation 28 exons on devise pair of primers and
One probe, used as internal reference, the primer and probe sequence are as follows：
E28-F:CGAGTATCTCAACACTGTCCAGC；
E28-R:GAAAGAAGTCCTGCTGGTAGTCAG；
TaqMan-MGB probes 2：CATTCGACAGCCCTGC.
The synthesis of above-mentioned primer and probe uses solid phase phosphoramidite triester method.Comprise the following steps that：
1) the blocking group DMT of solid phase carrier 5'- hydroxyls is removed with trichloroacetic acid, free 5'- hydroxyls are obtained；
2) phosphoramidite protection nucleotide monomer is mixed with activator tetrazole, is obtained in the middle of nucleosides phosphorous acid activation
, there is condensation reaction with free 5'- hydroxyls in body；
3) due to that cannot ensure that absolutely 5'- hydroxyls are involved in condensation, may have only a few 5'- hydroxyls not participate in
Reaction, can be terminated synthesis with acetic anhydride and 1- methylimidazole reagents, and this short-movie section can be separated in purifying；
4) in the presence of oxidant, phosphorous acyl form is changed into more stable phosphotriester, makes DNA phosphate backbones more steady
It is fixed.
Four steps more than a, deoxynucleotide is connected on solid phase carrier.Above step is repeated, until
The base connection of required synthesis is completed.
The Primer selection PAGE way of purification of all synthesis, TaqMan-MGB probes are in the end of primer 5 ' flag F AM, 3 ' ends
Mark MGB is obtained.Primer and 1 × TE of probe be diluted to 10 μM it is standby.
The method detected using primer of the invention is as follows：
1. the pretreatment of pair test sample：With conventional method rapid extraction sample DNA；
2. quantitative fluorescent PCR reaction system (20 μ L):
3.PCR response procedures：50 DEG C, 2min；95 DEG C of predegeneration 10min；40cycles:95℃,15sec、60℃30sec
(collection fluorescence).
4. 2 pipes of reaction point are carried out, and T790M detections are reacted and internal reference E28.
5. sample Ct (E28)<Result is effective when 30.If sample Ct (E28)>30, it should increase the amount of template, it is ensured that every
Template amount is at least 5ng in one pipe.△ Ct values are calculated according to formula △ Ct values=Ct (T790M)-Ct (E28) wild type；If △
Ct>9.0, it is judged to without mutation, if △ Ct≤9.0, it is judged to mutation.
Beneficial effects of the present invention：1. high accuracy：Precise Identification is mutated, and false positive rate is 0；2. quick, efficient amplification：
Amplification is completed by only needing 1 hour；3. sensitivity is high：Within reaching 10 copies to the lowest detection limit of EGFR gene；④
Identification is easy：By △ Ct value interpretations；5. purposes is wide, can be widely used for conventional detection and the epidemiology survey of clinic.
Brief description of the drawings
Fig. 1 is each sample testing result figure of the embodiment of the present invention.
Wherein, A is the testing result of sample 1, and B is the testing result of sample 2, and C is the testing result of sample 3, and D is sample
4 testing result.
Specific embodiment
Specific embodiment is provided below technical scheme is expanded on further, but technology application of the invention is not only limited
In embodiment.
Embodiment 1
1.T790M-F:CTCCACCGTGCAGCTCATCTT
2.T790M-R:TGCACACACCAGTTGAGCAGGT
3.TaqMan-MGB probes 1：TCGGCTGCCTCCTGGA
4.E28-F:CGAGTATCTCAACACTGTCCAGC
5.E28-R:GAAAGAAGTCCTGCTGGTAGTCAG
6.TaqMan-MGB probes 2：CATTCGACAGCCCTGC
The synthesis of above-mentioned primer and probe uses solid phase phosphoramidite triester method.Comprise the following steps that：
1) the blocking group DMT of solid phase carrier 5'- hydroxyls is removed with trichloroacetic acid, free 5'- hydroxyls are obtained；
2) phosphoramidite protection nucleotide monomer is mixed with activator tetrazole, is obtained in the middle of nucleosides phosphorous acid activation
, there is condensation reaction with free 5'- hydroxyls in body；
3) due to that cannot ensure that absolutely 5'- hydroxyls are involved in condensation, may have only a few 5'- hydroxyls not participate in
Reaction, can be terminated synthesis with acetic anhydride and 1- methylimidazole reagents, and this short-movie section can be separated in purifying；
4) in the presence of oxidant, phosphorous acyl form is changed into more stable phosphotriester, makes DNA phosphate backbones more steady
It is fixed.
Four steps more than a, deoxynucleotide is connected on solid phase carrier.Above step is repeated, until
The base connection of required synthesis is completed.
The Primer selection PAGE way of purification of all synthesis, TaqMan-MGB probes are in the end of primer 5 ' flag F AM, 3 ' ends
Mark MGB is obtained.Primer and 1 × TE of probe be diluted to 10 μM it is standby.
Embodiment 2：Quantitative fluorescent PCR quick detection EGFR gene T790M point mutation
Step one：Test sample is pre-processed
Conventionally extract sample DNA.
Wherein sample is as follows：
Sample 1：Remove the aseptic double-distilled water of DNAase
Sample 2：In the absence of the sample of EGFR gene T790M mutation
Sample 3：The cfDNA standard items of the 1%T790M frequencies of mutation
Sample 4：The cfDNA standard items of the 5%T790M frequencies of mutation
Step 2：Quantitative fluorescent PCR reaction system is configured
Quantitative fluorescent PCR reaction system (20 μ L):
T790M detections reaction and internal reference E28 reaction point 2 pipes configurations.Each sample will carry out this 2 reactions.
Step 3：Quantitative fluorescent PCR reaction is carried out
The reaction solution that will be configured in step 2 is reacted in Stepone Plus quantitative real time PCR Instruments by following procedure：50
DEG C, 2min；95 DEG C of predegeneration 10min；40cycles:95 DEG C, 15sec, 60 DEG C of 30sec (collection fluorescence).
Step 4：Result judgement
The result of sample 1 is shown in that Figure 1A, E28 and T790M reaction, without amplified signal, illustrate reaction system without DNA pollution.
The result of sample 2 is shown in Figure 1B, △ Ct=11.146>9.0, illustrate to be mutated in the absence of EGFR gene T790M.
The result of sample 3 is shown in Fig. 1 C, △ Ct=7.812<9.0, illustrate there is EGFR gene T790M mutation.
The result of sample 4 is shown in Fig. 1 D, △ Ct=5.464<9.0, illustrate there is EGFR gene T790M mutation.
Experimental data more than can be seen that laboratory test results and actually be consistent.By to testing result △ Ct values
Calculating, accurately EGFR gene T790M mutagenic samples can be identified.
Claims (2)
1. one group is used to detect the primer sets of EGFR gene T790M point mutation, it is characterised in that comprising following primer：
T790M-F:CTCCACCGTGCAGCTCATCTT；
T790M-R:TGCACACACCAGTTGAGCAGGT；
TaqMan-MGB probes 1：TCGGCTGCCTCCTGGA；
Wherein T790M-R is wild type anti-sense primer, and sense primer T790M-F is ARMS primers, is additionally drawn in 3 ' end seconds
Enter base mismatch T, make for wild-type template, there are 2 base mismatch, amplification efficiency is decreased obviously；And be mutated for T790M
Template, only one of which mispairing, and do not remain to specific amplification in 3 ' ends；So as to low in Sensitive Detection 5ng DNA samples
T790M to 1% is mutated.
2. one group as claimed in claim 1 is used to detect the primer sets of EGFR gene T790M point mutation, it is characterised in that
EGFR devises pair of primers and a probe on 28 exons without mutation, as internal reference, the primer and probe sequence
It is as follows：
E28-F:CGAGTATCTCAACACTGTCCAGC；
E28-R:GAAAGAAGTCCTGCTGGTAGTCAG；
TaqMan-MGB probes 2：CATTCGACAGCCCTGC.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107385066A (en) * | 2017-08-17 | 2017-11-24 | 浙江大学 | Detect primer pair, kit, detection method and the application of EGFR genetic mutation |
| CN108384852A (en) * | 2017-12-19 | 2018-08-10 | 上海百力格生物技术有限公司 | A kind of MGB primer and probe sequences for lung cancer EGFR gene T790M site primers |
| CN109355360A (en) * | 2018-11-20 | 2019-02-19 | 元码基因科技（北京）股份有限公司 | For detecting composition, kit and the method for EGFR mutation |
| CN109385463A (en) * | 2017-08-11 | 2019-02-26 | 武汉康昕瑞基因健康科技有限公司 | EGFR genetic mutation detection primer group, probe, kit and detection method |
| CN109628589A (en) * | 2018-11-20 | 2019-04-16 | 北京昱晟达医疗科技有限公司 | Detect primer, kit and the method for gene mutation |
| CN109929929A (en) * | 2017-12-15 | 2019-06-25 | 江苏众红生物工程创药研究院有限公司 | Kit and clinical application for Human epidermal growth factor receptor detection in Gene Mutation |
| CN109929930A (en) * | 2017-12-15 | 2019-06-25 | 江苏众红生物工程创药研究院有限公司 | Kit and clinical application for people's PIK3CA detection in Gene Mutation |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109385463A (en) * | 2017-08-11 | 2019-02-26 | 武汉康昕瑞基因健康科技有限公司 | EGFR genetic mutation detection primer group, probe, kit and detection method |
| CN107385066A (en) * | 2017-08-17 | 2017-11-24 | 浙江大学 | Detect primer pair, kit, detection method and the application of EGFR genetic mutation |
| CN107385066B (en) * | 2017-08-17 | 2021-03-23 | 浙江大学 | Primer pair, kit, detection method and application for detecting EGFR gene mutation |
| CN109929929A (en) * | 2017-12-15 | 2019-06-25 | 江苏众红生物工程创药研究院有限公司 | Kit and clinical application for Human epidermal growth factor receptor detection in Gene Mutation |
| CN109929930A (en) * | 2017-12-15 | 2019-06-25 | 江苏众红生物工程创药研究院有限公司 | Kit and clinical application for people's PIK3CA detection in Gene Mutation |
| CN108384852A (en) * | 2017-12-19 | 2018-08-10 | 上海百力格生物技术有限公司 | A kind of MGB primer and probe sequences for lung cancer EGFR gene T790M site primers |
| CN109355360A (en) * | 2018-11-20 | 2019-02-19 | 元码基因科技（北京）股份有限公司 | For detecting composition, kit and the method for EGFR mutation |
| CN109628589A (en) * | 2018-11-20 | 2019-04-16 | 北京昱晟达医疗科技有限公司 | Detect primer, kit and the method for gene mutation |
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