CN104830981A - KRAS gene multipoint mutation single tube rapid detection method and kit - Google Patents
KRAS gene multipoint mutation single tube rapid detection method and kit Download PDFInfo
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Abstract
The invention discloses a multipoint mutation single tube rapid detection method and a kit. The multipoint mutation single tube rapid detection method comprises the following steps: (1) designing an ARMS primer, a corresponding downstream primer, a medium connection primer, a common fluorescent probe, and a quenching probe of the common fluorescent probe; (2) mixing the ARMS primer, corresponding downstream primer, medium connection primer, common fluorescent probe and quenching probe of the common fluorescent probe with a hot-start rapid Taq enzyme system to carry out amplification; (3) detecting the fluorescence change of the reaction system to judging whether point mutation happens or not. The operation of the provided detection method is simple, and the existence of multipoint mutation can be determined qualitatively. The provided detection method has the advantages that the difficulty of primer design is lowered, thus the primer synthesis cost is largely reduced; the shortages of conventional ARMS technology are overcome, the detection flux is greatly increased; one reaction tube is adopted to replace the multi-tube detection, and the human and material resources are saved.
Description
Technical field
The present invention relates to a kind of mutation detection kit, particularly a kind of KRAS multipoint mutation single tube quick detection kit.
Background technology
KRAS gene is positioned on the mankind's No. 12 karyomit(e)s, is oncogene common in people's tumour.When KRAS gene is normal, the path of regulating cell growth can be controlled, can inhibition tumor cell growth.When undergoing mutation, Cellular Signaling Transduction Mediated can be caused disorderly, uncontrolled cellular proliferation and canceration.
Colorectal cancer (colorectal cancer, CRC) is one of malignant tumour that M & M is the highest in the world at present, and when the colorectal cancer patients of more than 40% is made a definite diagnosis, tumour has occurred to shift and lost the chance of radical surgery treatment.At present, molecular targeted therapy has become the common recognition of international and domestic metastatic colorectal carcinoma treatment.Monoclonal antibody drug Cetuximab (trade(brand)name Erbitux) as targeting EGFR has become the line medication in metastatic colorectal carcinoma treatment.
Patient due to KRAS wild-type is the maximum benefit crowd of anti-EGFR monoclonal antibody medicine treatment, the most sudden change of KRAS gene occurs on 2 exon 12,13 codons, so before the anti-EGFR monoclonal antibody medicine treatment of use, the abrupt climatic change of KRAS gene the 12nd and No. 13 codons is carried out in NCCN " colorectal cancer clinical practice guideline (Chinese version) " recommendation in 2011, thus based on the treatment plan that the mutation status of KRAS gene in patient tumors DNA instructs clinical formulation suitable.
The catastrophe point of KRAS genomic medicine sensitivity, generally believes at present and need detect 7 mutational sites, and fluorescence PCR detection reagent kit on the market generally uses 7 PCR pipe to detect, and analytic process is loaded down with trivial details and there is no need.Whole testing process is comparatively slow simultaneously, at least needs within 2 days, just can go out result.
This patent uses multipoint mutation Parallel detection, in single tube, by the indicative function of fluorophor, in same PCR reaction, can detect multiple mutational site simultaneously.Separately several PCR pipe is not needed to carry out respectively.Realize simple, KRAS point mutation detection easily.
Summary of the invention
The object of the present invention is to provide a kind of KRAS multipoint mutation single tube method for quick and test kit efficiently.
The technical solution used in the present invention is:
Multipoint mutation single tube method for quick, comprises the steps:
1) downstream primer, the intermediary that design ARMS primer and the correspondence of suddenling change for difference connect primer, universal fluorescent probe and quenching probes thereof; Wherein, intermediary connects primer and is made up of universal sequence part and distinguished sequence part, universal sequence part and universal fluorescent probe portion complementary, the partial sequence complementarity in distinguished sequence part and point mutation downstream;
2) downstream primer of ARMS primer and correspondence, intermediary are connected primer, universal fluorescent probe and quenching probes thereof and mix with the quick Taq enzyme system of warm start, increase;
3) change in fluorescence of detection reaction system, determines whether there is point mutation.
It is excellent in degeneracy that downstream primer, intermediary that ARMS primer pair is answered connect primer, universal fluorescent probe, namely downstream primer, intermediary connects primer, universal fluorescent probe can correspond to many.
As the further improvement of aforesaid method, Nucleotide universal fluorescent probe connecting fluorophor is no more than 4 Nucleotide with the spacing of Nucleotide after complementary pairing quenching probes being connected quenching group, further, 3,2,1 or be connected on two Nucleotide of mutually pairing is no more than.
As the further improvement of aforesaid method, intermediary connects in primer and is no less than 5 bp with the nucleic acid quantity of the partial sequence complementarity in downstream, mutational site, is no less than 10 bp, and especially, complementary nucleic acid quantity is 15 ~ 30bp.
As the further improvement of aforesaid method, intermediary connects the nucleic acid quantity of matching with universal fluorescent probe portion complementary nucleic acid in primer and is no less than 5 bp, is no less than 10 bp, and especially, complementary nucleic acid quantity is 15 ~ 45bp.
The existence of complementary sequence, is conducive to ensureing to there is enough avidity between primer, ensures the reliability of detected result.
As the further improvement of aforesaid method, one end of universal fluorescent probe is connected with protection nucleotide sequence.The effect of protection sequence is to prevent the critical sequences of probe from not excised too early by the exonuclease being not intended in reaction system introduce.Protection sequence not with the complementary pairing such as sequence to be measured, primer, can be simple repeated sequence, as multiple A, T, C, G etc.
A kind of KRAS multipoint mutation single tube quick detection kit, comprise DNA extracting solution, the quick Taq enzyme system of warm start and KRAS detect primer, it is characterized in that: described KRAS detect primer by organizing the downstream primer of ARMS primer and correspondence more, intermediary connects primer, universal fluorescent probe and quenching probes thereof and forms; Wherein: intermediary connects a terminal sequence of primer and treats the partial sequence complementarity of KRAS sites downstream, and the other end sequence and universal fluorescent probe portion complementary nucleic acid match.
Preferably, the intermediary used in above-mentioned KRAS multipoint mutation single tube quick detection kit connects in primer and is no less than 5 bp with the nucleic acid quantity of the partial sequence complementarity of KRAS sites downstream, be no less than 10 bp, especially, complementary nucleic acid quantity is 15 ~ 30bp.
Preferably, the intermediary used in above-mentioned KRAS multipoint mutation single tube quick detection kit connects the nucleic acid quantity of matching with universal fluorescent probe portion complementary nucleic acid in primer and is no less than 5 bp, is no less than 10 bp, and especially, complementary nucleic acid quantity is 15 ~ 45bp.
Especially, the sequence of the ARMS primer used in above-mentioned KRAS multipoint mutation single tube quick detection kit is as follows:
ARMS primer 1:ACTTGTGGTAGTTGGAGCTGA
ARMS primer 2: ACTTGTGGTAGTTGGAGCTGC
ARMS primer 3:ACTTGTGGTAGTTGGAGCTGT
ARMS primer 4:ACTTGTGGTAGTTGGAGCTA
ARMS primer 5:ACTTGTGGTAGTTGGAGCTC
ARMS primer 6:ACTTGTGGTAGTTGGAGCTT
ARMS primer 7:GTGGTAGTTGGAGCTGGTGA
The downstream primer of ARMS primer 1 ~ 7 correspondence is Y1:ATTAAAACAAGATTTACCTCTA;
Intermediary's primer of ARMS primer 1 ~ 7 correspondence is Z1:
GTCAGCCTTGCTAAGGCTACAGCTAATTCAGAATCA。
Especially, the sequence of the universal fluorescent probe used in above-mentioned KRAS multipoint mutation single tube quick detection kit is:
CT (dA-FAM) GAGCTCTACGTAGCCTTAGCAAGGCTGACTTTTTTTTTTTTTTTTTT, corresponding cancellation sequence is: 5 ' BHQ-GCTCTAG-3 '.
Warm start quick Taq enzyme system consist of dNTPS(U), UNG enzyme, the quick Taq enzyme of warm start.
The invention has the beneficial effects as follows:
The present invention is simple to operate, qualitative detection can go out the existence of multipoint mutation.The design of primers difficulty used in detection method of the present invention is low, significantly reduces the cost of primer synthesis.
Detection method overcomes the limitation of existing ARMS technology, substantially increases it and detects flux.
Detection method of the present invention achieves the simplification of multitube detection reaction, and the multitube before 1 tube reaction instead of detects, and uses manpower and material resources sparingly.
Quick Taq enzyme is used in detection method of the present invention, better than general T aq enzyme.
Accompanying drawing explanation
Fig. 1 to Fig. 6 is the schematic diagram of detection method;
Fig. 7 is the pcr amplification curve of isoconcentration template under different Taq enzyme reaction conditions, illustrates that the quick Taq enzyme used in this test kit is better than the expanding effect of general T aq enzyme.
Embodiment
Below in conjunction with accompanying drawing, further illustrate Cleaning Principle of the present invention.
For KRAS point 12Asp, the wild-type of this point is G, and saltant type is A, in PCR process, first uses 1 pair of primer, containing 1 ARMS primer for catastrophe point (last base of primer is A) and downstream primer.This primer is specific under the effect of quick enzyme amplifies mutational site A.Wild type site G is not amplified, as shown in Figure 1;
After specific amplification occurs, under the guiding of ARMS primer, quick Taq enzyme along DNA profiling, to 5 '-3 ' direction moves and is hydrolyzed intermediary and connect the sequence mediates region of primer, the probes complementary region that intermediary connects primer is then released, as shown in Figure 2 and Figure 3;
The probes complementary region discharged, based on base pair complementarity principle, with universal fluorescent probe, complementation occurs, under the existence of quenching group, the fluorescence of fluorophor is quenched, as shown in Figure 4;
Quick Taq enzyme connects the probes complementary region of primer, to 5 '-3 by intermediary ' direction moves, hydrolysis quenching group, thus quenching group is away from fluorophor, thus PCR reaction creates fluorescence, as shown in Figure 5, Figure 6.
Therefore, as long as the ARMS primer in multiple mutational sites, downstream primer are connected primer with intermediary, 1 group of universal fluorescent probe and general cancellation complementary probe, can be placed in a PCR reaction tubes, namely can detect in sample whether there is point mutation, significantly improve the detection flux of ARMS method.
Embodiment 1
1. KRAS multipoint mutation single tube quick detection kit, principal constituent is as follows:
1) DNA extraction liquid:
50mM NaOH, 10mMTris-HCl(PH8.0), 1%NP-40,6%Chelex, 0.1mM EDTA(PH8.0) composition
2) quick Taq enzyme system:
The dNTPs(25mM of quick Taq, the 0.5ul of 3U)
3) primer:
The downstream primer of KRAS ARMS primer and correspondence:
| Codon | Mutant designations | ARMS primer sequence (5 ' → 3 ') | Downstream primer sequence (5 ' → 3 ') |
| 12 | G12D (Gly12Asp) | ACTTGTGGTAGTTGGAGCTGA | ATTAAAACAAGATTTACCTCTA |
| 12 | G12A (Gly12Ala) | ACTTGTGGTAGTTGGAGCTGC | ATTAAAACAAGATTTACCTCTA |
| 12 | G12V (Gly12Val) | ACTTGTGGTAGTTGGAGCTGT | ATTAAAACAAGATTTACCTCTA |
| 12 | G12S (Gly12Ser) | ACTTGTGGTAGTTGGAGCTA | ATTAAAACAAGATTTACCTCTA |
| 12 | G12R (Gly12Arg) | ACTTGTGGTAGTTGGAGCTC | ATTAAAACAAGATTTACCTCTA |
| 12 | G12C (Gly12Cys) | ACTTGTGGTAGTTGGAGCTT | ATTAAAACAAGATTTACCTCTA |
| 13 | G13D (Gly13Asp) | GTGGTAGTTGGAGCTGGTGA | ATTAAAACAAGATTTACCTCTA |
Intermediary connects primer:
4) universal fluorescent probe is to as follows:
Universal fluorescent probe: 5 '
-CT (dA-FAM) GAGCtCTACGT
aGCCTTAGCAAGGCTGACtTTTTTTTTTTTTTTTTT-3 ',
General cancellation complementary probe:
5 ' BHQ-GCTCTAG-3 '.
2. use KRAS multipoint mutation single tube quick detection kit, operation steps is as follows:
1) DNA extraction:
Get the FFPE tissue slice of 2 10 μm, put into after scraping down in 1.5ml centrifuge tube, then add the DNA extraction liquid of 120 μ l, 100 DEG C boil 10 minutes after, by centrifugal for centrifuge tube 12000rpm 5 minutes, the supernatant getting 10 μ l joined in PCR reaction tubes.
2) fast PCR:
In 50 μ l fluorescent PCR systems, the quick Taq enzyme of 3U, the UNG enzyme of 10mM Tris-HCL, 50mM KCL, 0.5U, 0.2mM dNTPS(U), 3mM MgCl2.Universal fluorescent probe and each 1 μm of general cancellation complementary probe, the ARMS primer in 28 mutational sites, downstream primer are connected each 1 μm of primer with intermediary.After 95 degree of 5min, 95 DEG C 5 seconds, 58 DEG C 31 seconds, cycle number is 40 circulations.
The change in fluorescence of monitoring reaction system, as there is point mutation, then fluorescent signal can increase.
<110> Guangzhou two kinds of substance synthesis into another Technology Co., Ltd.
<120> KRAS gene multipoint mutation single tube method for quick and test kit
<130>
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
The artificial primer of <213>
<400> 1
acttgtggta gttggagctg a 21
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<211> 21
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The artificial primer of <213>
<400> 2
acttgtggta gttggagctg c 21
<210> 3
<211> 21
<212> DNA
The artificial primer of <213>
<400> 3
acttgtggta gttggagctg t 21
<210> 4
<211> 20
<212> DNA
The artificial primer of <213>
<400> 4
acttgtggta gttggagcta 20
<210> 5
<211> 20
<212> DNA
The artificial primer of <213>
<400> 5
acttgtggta gttggagctc 20
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<211> 20
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The artificial primer of <213>
<400> 6
acttgtggta gttggagctt 20
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<211> 20
<212> DNA
The artificial primer of <213>
<400> 7
gtggtagttg gagctggtga 20
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<211> 22
<212> DNA
The artificial primer of <213>
<400> 8
attaaaacaa gatttacctc ta 22
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<212> DNA
The artificial primer of <213>
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gtcagccttg ctaaggctac agctaattca gaatca 36
<210> 10
<211> 47
<212> DNA
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gagctctacg tagccttagc aaggctgact tttttttttt ttttttt 47
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<213> general probe
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agccttagca aggctgac 18
Claims (9)
1. multipoint mutation single tube method for quick, comprises the steps:
A) downstream primer, the intermediary that design ARMS primer and the correspondence of suddenling change for difference connect primer and universal fluorescent probe, wherein, intermediary connects primer by the universal sequence part with universal fluorescent probe portion complementary with form with the specific part of the partial sequence complementarity in point mutation downstream; B) by the downstream primer of ARMS primer and correspondence, intermediary connects primer and universal fluorescent probe mixes with quick Taq enzyme system, increase; C) change in fluorescence of detection reaction system, determines whether there is point mutation.
2. a KRAS multipoint mutation single tube quick detection kit, comprise DNA extraction liquid, quick Taq enzyme system and KRAS detect primer, it is characterized in that: described KRAS detects primer by downstream primer, the intermediary's connection primer of organizing ARMS primer and correspondence more, and universal fluorescent probe composition, wherein:
Intermediary connects a terminal sequence of primer and treats the partial sequence complementarity of KRAS sites downstream, and the other end sequence and universal fluorescent probe portion complementary nucleic acid match.
3. KRAS multipoint mutation single tube quick detection kit according to claim 2, is characterized in that: fast Taq enzyme system consist of dNTPS(U), UNG enzyme, fast Taq enzyme.
4. KRAS multipoint mutation single tube quick detection kit according to claim 2, is characterized in that: intermediary connects in primer and is no less than 5 bp with the nucleic acid quantity of the partial sequence complementarity of KRAS sites downstream.
5. KRAS multipoint mutation single tube quick detection kit according to claim 4, is characterized in that: it is 15 ~ 30bp that intermediary to connect in primer with the nucleic acid quantity of the partial sequence complementarity of KRAS sites downstream.
6. KRAS multipoint mutation single tube quick detection kit according to claim 2, is characterized in that: intermediary connects the nucleic acid quantity of matching with universal fluorescent probe portion complementary nucleic acid in primer and is no less than 5 bp.
7. KRAS multipoint mutation single tube quick detection kit according to claim 6, is characterized in that: it is 15 ~ 45bp that intermediary connects the nucleic acid quantity of matching with universal fluorescent probe portion complementary nucleic acid in primer.
8. the KRAS multipoint mutation single tube quick detection kit according to claim 2 ~ 7 any one, is characterized in that: the sequence of ARMS primer is as follows:
。
9. KRAS multipoint mutation single tube quick detection kit according to claim 8, is characterized in that: the sequence of universal fluorescent probe is:
5 '
-CT (dA-FAM) GAGCtCTACGT
aGCCTTAGCAAGGCTGACtTTTTTTTTTTTTTTTTT-3 ', corresponding cancellation sequence is: 5 ' BHQ-GCTCTAG-3 '.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105803088A (en) * | 2016-04-29 | 2016-07-27 | 广州市康立明生物科技有限责任公司 | Prime group, probe group and kit for detecting Kras gene mutation |
| CN106868127A (en) * | 2017-02-20 | 2017-06-20 | 人和未来生物科技（长沙）有限公司 | People's KRAS gene multipoint mutation single tube joint inspections fluorescence PCR method, kit and system |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105803088A (en) * | 2016-04-29 | 2016-07-27 | 广州市康立明生物科技有限责任公司 | Prime group, probe group and kit for detecting Kras gene mutation |
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| CN108060215A (en) * | 2018-02-08 | 2018-05-22 | 天津安必森生物技术有限公司 | A kind of kit for detecting people's KRAS gene mutation |
| CN112795653A (en) * | 2021-02-08 | 2021-05-14 | 深圳市核子基因科技有限公司 | Primer probe set and kit for KRAS gene mutation detection and application thereof |
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