CN104531889A - Improved amplification refractory mutation detection system primer and application thereof - Google Patents
Improved amplification refractory mutation detection system primer and application thereof Download PDFInfo
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- CN104531889A CN104531889A CN201510023443.7A CN201510023443A CN104531889A CN 104531889 A CN104531889 A CN 104531889A CN 201510023443 A CN201510023443 A CN 201510023443A CN 104531889 A CN104531889 A CN 104531889A
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Abstract
The invention relates to the field of molecular biology and in particular relates to an improved amplification refractory mutation detection system primer and application thereof. The improved amplification refractory mutation detection system primer comprises an upstream primer and a downstream primer, wherein 2-3 bases not paired with a target sequence are introduced to the 5' terminal of the upstream primer, and a mismatched base is introduced to the antepenult base position of the 3' terminal of the upstream primer. The invention also provides the application of the improved amplification refractory mutation detection system primer in an amplification refractory mutation detection system. According to the improved amplification refractory mutation detection system primer and the application therefore, the sensitivity and specificity of the ARMS primers can be effectively improved.
Description
Technical field
The present invention relates to biology field, particularly relate to a kind of amplification Refracting Mutation detection system primer and application of improvement.
Background technology
Lung cancer is one of current modal malignant tumour, is also one of cancer of high lethality rate, and wherein the patient of 80-85% is nonsmall-cell lung cancer (non-small cell lung cancers, NSCLC).In recent years along with the raising of molecular biology level, create the antitumor drug for molecular target.These medicines are with strong points, killing tumor cell that can be special, Be very effective.Wherein with EGF-R ELISA (Epidermal growth factor receptor, EGFR) for the tyrosine kinase inhibitor of target spot is outstanding day by day in Treatment for Non-small Cell Lung.EGFR is as the target spot of cancer therapy, targeted drug mainly contains two classes: a class is that the small molecule tyrosine kinase inhibitors (tyrosine kinase inhibitor, TKI) acted in acceptor born of the same parents mainly comprises Gefitinib, erlotinib, EKB-569, PKI-166, GW-2016 and CI-1033; Another kind of is the monoclonal antibody (MAb) acting on receptor extracellular region, comprises Cetuximab (cetuximab), ABX-EGF and EMD72000.These medicines suppress its phosphorylation and intracellular signaling by the activity blocking EGFR, thus play antitumor action, also can increase the antitumor curative effect of chemotherapy and radiation simultaneously.
Large quantity research shows, and not all patients with lung cancer benefits from TKI treatment, and EGFR sudden change is relevant to TKI curative effect.The NSCLC patient carrying EGFR genetic mutation is evident in efficacy when accepting Iressa and Erlotinib treatment, but research finds T790M site on EGFR extron 20 and is inserted as resistance site, can suppress the curative effect of medication such as Iressa and Erlotinib.These sudden changes of clinical detection can predict TKI result for the treatment of well, for tumour medication provides guidance foundation.
Amplification Refracting Mutation detection system (Amplification refractory mutation system, ARMS), also known as allele specific pcr (Allele-specific PCR, AS-PCR), be mutation detection techniques most widely used at present, its principle utilizes Taq enzyme disappearance proofreading activity to complete the detection of sudden change.In the reaction system of ARMS, primer marking 2 different fluoresceins or adding the methods such as fluorescent probe is all methods of genotyping based on real-time fluorescence PCR.Can detect that in sample, content is low to moderate the mutator gene of 0.1-1.0%.At present, the method has become one of most important, state-of-the-art technology of tumour individuation Molecular Detection in the world, and its advantage in clinical application is extensively approved by expert.During ARMS design of primers, usually play the 2nd base positions at 3 '-end and add mispairing, to suppress the amplification of wild-type template, therefore ARMS has good SNP detectivity; But the shortcoming of ARMS technology is that susceptibility is lower, the sudden change of 10 copy left and right can only be detected.
Summary of the invention
For solving the problems of the technologies described above, the object of this invention is to provide a kind of amplification Refracting Mutation detection system primer of improvement of Sensitivity and Specificity of effective raising ARMS primer.
The amplification Refracting Mutation detection system primer of improvement of the present invention, comprise upstream primer and downstream primer, wherein, 5 ' end of upstream primer introduces 2-3 base of not matching with target sequence, and described upstream primer 3 ' end third from the bottom base positions introduces base mismatch.
Further, the base that described upstream primer 5 ' end is introduced is GG, CC or CG.
Further, the base mismatch of described upstream primer 3 ' end be strong base mismatch and in strong base mismatch, described strong base mismatch comprises C-T and G-A, and in described, strong base mismatch comprises A-A, G-G and C-C.
Another object of the present invention is to provide a kind of application of amplification Refracting Mutation detection system primer in amplification Refracting Mutation detection system of improvement.
Another object of the present invention is to provide a kind of primer, EGFR21 exon L858R G>T sudden change is detected for adopting amplification Refracting Mutation detection system, described primer comprises upstream primer and downstream primer, 5 ' end of described upstream primer introduces 2-3 base of not matching with target sequence, and described upstream primer 3 ' end third from the bottom base positions introduces base mismatch.
Further, the base that described upstream primer 5 ' end is introduced is GG, CC or CG.
Further, the base mismatch of described upstream primer 3 ' end be strong base mismatch and in strong base mismatch, described strong base mismatch comprises C-T and G-A, and in described, strong base mismatch comprises A-A, G-G and C-C.
Further, described upstream primer is for having the upstream primer of sequence shown in SEQ ID NO:1, and described downstream primer is for having the downstream primer of sequence shown in SEQ ID NO:4.
Another object of the present invention is to provide a kind of test kit detecting EGFR21 exon L858R G>T and suddenly change, and it is characterized in that: comprise the upstream primer with SEQ ID NO:1 and the downstream primer with sequence shown in SEQ ID NO:4.
Further, also comprise the Taqman probe with SEQ ID NO:5,5 ' end and the 3 ' end of described Taqman probe are marked by FAM and BHQ1 respectively.
By such scheme, the present invention at least has the following advantages: the 5 ' end due to upstream primer introduces 2-3 base of not matching with target sequence, after amplification 2-3 circulation, " GG ", " CC " and " CG " sequence that 5 ' end is introduced then becomes the template of lower whorl amplification.The advantage of such design: the bonding force that improve primer and template, improves the susceptibility of ARMS primer.In addition, because upstream primer 3 ' end third from the bottom base positions introduces base mismatch, in the process of PCR, the amplification of wild-type template is suppressed.
Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to better understand technique means of the present invention, and can be implemented according to the content of specification sheets, coordinates accompanying drawing to be described in detail as follows below with preferred embodiment of the present invention.
Accompanying drawing explanation
Fig. 1 is principle schematic of the present invention.
Embodiment
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in further detail.Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment one
As shown in Figure 1, the amplification Refracting Mutation detection system primer of improvement of the present invention, comprises upstream primer and downstream primer, wherein, 5 ' end of upstream primer introduces 2-3 base of not matching with target sequence, and upstream primer 3 ' end third from the bottom base positions introduces base mismatch.
5 ' the end due to upstream primer introduces 2-3 base of not matching with target sequence, and after amplification 2-3 circulation, " GG ", " CC " and " CG " sequence that 5 ' end is introduced then becomes the template of lower whorl amplification.Because upstream primer 3 ' end third from the bottom base positions introduces base mismatch, in the process of PCR, suppress the amplification of wild-type template.
Embodiment two
EGFR 21 exon L858R G>T abrupt climatic change.
1, the preparation of L858R standard substance.The order-checking screening L858R positive and ' negative ' specimens, be cloned in pGEM-T carrier, be built into recombinant plasmid, be converted in escherichia coli DH5a, extract recombinant plasmid, order-checking qualification, diluted for use.
2, primed probe design: common ARMS primer and application this patent design primer are in table 1, and with Taqman probe for signalling system carries out fluoroscopic examination, primer is sent to the raw work synthesis in Shanghai.
Table 1 detects EGFR 21 exon L858R primed probe
What 5 ' end of upstream primer (F1) introduced CG sequence (italic underlines mark) is 5 '-sequence introduced of end; Second from the bottom or the 3rd base positions of 3 ' end of upstream primer (F1, F2 and F3) introduces mispairing (underscore places large overstriking mark); 3 ' end of upstream primer (F1, F2 and F3) is mutating alkali yl (amplifying overstriking mark).
3, positive criteria product are detected.The ARMS of application this patent design detects 1E+5,1E+4 respectively and copies wild plasmid, and 1E+4,1E+3,1E+2,1E+1 and 5 copy mutant plasmids, PCR negative control water replaces.Identical system and condition as follows:
1) 25ul PCR system system is as follows:
2) reaction conditions is: 94 DEG C of denaturations 10 minutes; 40 circulations, 94 DEG C of sex change 30 seconds, 60 DEG C of annealing 45 seconds, 72 DEG C extend 25 seconds.Application ABI 7500 fluorescent PCR instrument annealing steps detects fluorescence, and after amplification terminates, data analysis pattern is " Auto " automatic analysis.
3) interpretation of result: ARMS primer specificity and the susceptibility of the design of application this patent are higher; Traditional ARMS primer introduces the strong mispairing of A/G in 3 '-end, second position, although can improve specificity, susceptibility is very low, can only detect the plasmid of 1E+2 copy; Introduce weak mispairing, although susceptibility is improved, specificity is poor.In the present invention, ARMS primer can detect the mutagenesis template of 5 copies (see table 2.Data in table are Ct value).
Table 2ARMS detects negative and positive plasmid
| Sample | F1 | F2 | F3 |
| W-1E+5copies | 36.21 | 32.53 | NA |
| W-1E+4copies | NA | 36.51 | NA |
| M-1E+4copies | 24.01 | 22.84 | 31.01 |
| M-1E+3copies | 27.34 | 26.26 | 34.56 |
| M-1E+2copies | 30.85 | 29.89 | 38.45 |
| M-1E+1copies | 35.09 | 34.74 | NA |
| M-5copies | 37.39 | 36.24 | NA |
| Water | NA | NA | NA |
4, the ARMS applying the present invention's design detects hybrid template.The ARMS applying the present invention's design detects 1E+4 copy mixing plasmid, contains the L858R mutagenesis template of 10%, 1%, 0.1%, 0.5% respectively.The ARMS of the present invention's design, can detect the mutant plasmid of 0.05%, higher than common ARMS from the mixing plasmid of 1E+4.
5, clinical sample detect, 20 routine clinical samples, application the present invention design ARMS detect, detected result and sequencing result completely the same.
The above is only the preferred embodiment of the present invention; be not limited to the present invention; should be understood that; for those skilled in the art; under the prerequisite not departing from the technology of the present invention principle; can also make some improvement and modification, these improve and modification also should be considered as protection scope of the present invention.
Claims (10)
1. the amplification Refracting Mutation detection system primer of an improvement, it is characterized in that: comprise upstream primer and downstream primer, wherein, 5 ' end of upstream primer introduces 2-3 base of not matching with target sequence, and described upstream primer 3 ' end third from the bottom base positions introduces base mismatch.
2. the amplification Refracting Mutation detection system primer of improvement according to claim 1, is characterized in that: the base that described upstream primer 5 ' end is introduced is GG, CC or CG.
3. the amplification Refracting Mutation detection system primer of improvement according to claim 1, it is characterized in that: the base mismatch of described upstream primer 3 ' end be strong base mismatch and in strong base mismatch, described strong base mismatch comprises C-T and G-A, and in described, strong base mismatch comprises A-A, G-G and C-C.
4. the application of amplification Refracting Mutation detection system primer in amplification Refracting Mutation detection system of the improvement described in any one of claims 1 to 3.
5. a primer, EGFR21 exon L858R G>T sudden change is detected for adopting amplification Refracting Mutation detection system, it is characterized in that: described primer comprises upstream primer and downstream primer, 5 ' end of described upstream primer introduces 2-3 base of not matching with target sequence, and described upstream primer 3 ' end third from the bottom base positions introduces base mismatch.
6. primer according to claim 5, is characterized in that: the base that described upstream primer 5 ' end is introduced is GG, CC or CG.
7. primer according to claim 5, is characterized in that: the base mismatch of described upstream primer 3 ' end be strong base mismatch and in strong base mismatch, described strong base mismatch comprises C-T and G-A, and in described, strong base mismatch comprises A-A, G-G and C-C.
8. primer according to claim 5, is characterized in that: described upstream primer is for having the upstream primer of sequence shown in SEQ ID NO:1, and described downstream primer is for having the downstream primer of sequence shown in SEQ ID NO:4.
9. detect the test kit that EGFR21 exon L858R G>T suddenlys change, it is characterized in that: comprise the upstream primer with SEQ ID NO:1 and the downstream primer with sequence shown in SEQ ID NO:4.
10. the test kit of detection EGFR21 exon L858R G>T according to claim 5 sudden change, it is characterized in that: also comprise the Taqman probe with SEQ ID NO:5,5 ' end and the 3 ' end of described Taqman probe are marked by FAM and BHQ1 respectively.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755551A (en) * | 2017-03-20 | 2017-05-31 | 陈超 | A kind of EGFR/L858R is mutated liquid biopsy kit and its application |
| CN107937572A (en) * | 2018-01-17 | 2018-04-20 | 福建农林大学 | The detection method of the anti-chalk disease associated SNP positions C2587245T of honeybee |
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2015
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755551A (en) * | 2017-03-20 | 2017-05-31 | 陈超 | A kind of EGFR/L858R is mutated liquid biopsy kit and its application |
| CN107937572A (en) * | 2018-01-17 | 2018-04-20 | 福建农林大学 | The detection method of the anti-chalk disease associated SNP positions C2587245T of honeybee |
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Application publication date: 20150422 |