CN106148521B - A kind of multipoint mutation single tube rapid detection method and kit - Google Patents
A kind of multipoint mutation single tube rapid detection method and kit Download PDFInfo
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Abstract
The invention discloses a kind of multipoint mutation single tube rapid detection method and kits.Multipoint mutation single tube rapid detection method, comprising: 1) design ARMS primer and corresponding downstream primer, intermediary's connection primer, universal fluorescent probe and its quenching probes；2) ARMS primer and corresponding downstream primer, intermediary's connection primer, universal fluorescent probe and its quenching probes are mixed with the quick Taq enzyme system of thermal starting, is expanded；3) change in fluorescence of reaction system is detected, it is determined whether there are point mutation.Invention of the invention, it is easy to operate, the presence of multipoint mutation can be gone out with qualitative detection.Design of primers difficulty used in detection method of the invention is low, significantly reduces the cost of primer synthesis；The limitation for overcoming existing ARMS technology substantially increases it and detects flux；It realizes 1 tube reaction to detect instead of multitube before, use manpower and material resources sparingly.
Description
Technical field
The present invention relates to a kind of mutation detection kit, in particular to a kind of multipoint mutation single tube quick detection kit.
Background technique
Point mutation and many tumours have close relationship, and existing research shows that the point mutation in certain group sites can make
The incidence of tumour greatly improves.By taking EGF-R ELISA (EGFR) as an example, EGFR is a kind of sugar of people's cell film surface
Protein receptor, the gene are located at No. 7 chromosomes of the mankind, are made of 28 exons.EGFR has tyrosine kinase activity, is former
The expression product of oncogene c-erbB1, tyrosine kinase functional areas are encoded by exons 1 8-24.EGFR mainly passes through RAS-
Extracellular signal is converted intracellular signal by RAF-MEK-ERK-MAPK signal path and PI3K-PDK signal path, thus to cell
Growth, proliferation, differentiation and apoptosis generation effect.
Lung cancer is the highest tumour of China's morbidity and mortality, wherein non-small cell lung cancer (non-small cell
Lung cancer, NSCLC) account for about the 80% of all lung cancer, and 50% or more there are EGFR gene bodies in China NSCLC patient
Cell mutation.It is existing research shows that EGFR genetic mutation is there are diversified forms, fractional mutations and EGFR tyrosine kinase inhibitor
If Gefitinib is effective related to Tarceva, fractional mutations are related to the drug resistance of EGFR tyrosine kinase inhibitor.Therefore,
Before carrying out targeted therapy using tyrosine kinase inhibitor, NCCN " non-small cell lung cancer clinical practice guideline (China in 2011
Version) " recommend to carry out EGFR genetic mutation detection, to instruct to face based on the mutation status of EGFR gene in patient tumors DNA
Bed formulates suitable therapeutic scheme.
Since the catastrophe point of EGFR gene medicaments insensitive is numerous, common is as shown in the table:
28 mutational sites need to be detected by generally believing at present.Existing fluorescence PCR detection reagent kit on the market generally uses 7
A PCR pipe is detected, and analytic process is cumbersome and is not necessarily to.Entire testing process is slower simultaneously, at least needs 2 days ability
Result out.
It is especially a kind of more quick it is necessary to develop a kind of detection kit of multipoint mutation more rapidly and efficiently
The detection kit of efficient EGFR multipoint mutation.
Summary of the invention
The purpose of the present invention is to provide a kind of efficient multipoint mutation single tube rapid detection method and EGFR multipoint mutations
Single tube quick detection kit.
The technical solution used in the present invention is:
Multipoint mutation single tube rapid detection method, includes the following steps:
1) design connects primer, universal fluorescent for the ARMS primer of different point mutation and corresponding downstream primer, intermediary
Probe and its quenching probes；Wherein, intermediary's connection primer is made of universal sequence part and distinguished sequence part, universal sequence portion
Divide, the partial sequence complementarity of distinguished sequence part and point mutation downstream complementary with universal fluorescent probe portion sequence；
2) by ARMS primer and corresponding downstream primer, intermediary's connection primer, universal fluorescent probe and its quenching probes with
The quick Taq enzyme system mixing of thermal starting, amplification；
3) change in fluorescence of reaction system is detected, it is determined whether there are point mutation.
The corresponding downstream primer of ARMS primer, intermediary's connection primer, universal fluorescent probe are excellent for degeneracy, i.e. a downstream
Primer, intermediary's connection primer, universal fluorescent probe can correspond to a plurality of.
As the further improvement of the above method, the nucleotide and quenching probes of fluorophor are connected on universal fluorescent probe
Spacing of the nucleotide of upper connection quenching group after complementary pairing is no more than 4 nucleotide, further, no more than 3,2
It is a, 1 or be connected on two nucleotide being mutually paired.
As the further improvement of the above method, intermediary connect in primer with the partial sequence complementarity in mutational site downstream
Nucleic acid quantity is no less than 5bp, no less than 10bp, and particularly, complementary nucleic acid quantity is 15~30bp.
As the further improvement of the above method, intermediary connects in primer and matches with universal fluorescent probe portion complementary nucleic acid
Nucleic acid quantity be no less than 5bp, no less than 10bp, particularly, complementary nucleic acid quantity be 15~45bp.
As the further improvement of the above method, one end of universal fluorescent probe is connected with protection nucleic acid sequence.
The beneficial effects of the present invention are:
Invention of the invention, it is easy to operate, the presence of multipoint mutation can be gone out with qualitative detection.In detection method of the invention
The design of primers difficulty used 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 invention realizes the simplification of multitube detection reaction, and 1 tube reaction is examined instead of multitube before
It surveys.It uses manpower and material resources sparingly.
Detailed description of the invention
Fig. 1 is the schematic diagram of detection method；
Fig. 2 is PCR amplification curve of the isoconcentration template under different Taq enzyme reaction conditions.
Specific embodiment
Multipoint mutation single tube rapid detection method, includes the following steps:
1) design connects primer, universal fluorescent for the ARMS primer of different point mutation and corresponding downstream primer, intermediary
Probe and its quenching probes；Wherein, intermediary's connection primer is made of universal sequence part and distinguished sequence part, universal sequence portion
Divide, the partial sequence complementarity of distinguished sequence part and point mutation downstream complementary with universal fluorescent probe portion sequence；
2) by ARMS primer and corresponding downstream primer, intermediary's connection primer, universal fluorescent probe and its quenching probes with
The quick Taq enzyme system mixing of thermal starting, amplification；
3) change in fluorescence of reaction system is detected, it is determined whether there are point mutation.
As the further improvement of the above method, the nucleotide and quenching probes of fluorophor are connected on universal fluorescent probe
Spacing of the nucleotide of upper connection quenching group after complementary pairing is no more than 4 nucleotide, further, no more than 3,2
It is a, 1 or be connected on two nucleotide being mutually paired.Universal fluorescent probe and quenching probes are also possible to have in same
There is the nucleic acid sequence of hairpin structure.
As the further improvement of the above method, intermediary connect in primer with the partial sequence complementarity in mutational site downstream
Nucleic acid quantity is no less than 5bp, no less than 10bp, and particularly, complementary nucleic acid quantity is 15~30bp.
As the further improvement of the above method, intermediary connects in primer and matches with universal fluorescent probe portion complementary nucleic acid
Nucleic acid quantity be no less than 5bp, no less than 10bp, particularly, complementary nucleic acid quantity be 15~45bp.
The presence of complementary series advantageously ensures that there are enough affinity between primer, guarantee the reliability of testing result.
As the further improvement of the above method, one end of universal fluorescent probe is connected with protection nucleic acid sequence.Protect sequence
The effect of column is to prevent the critical sequences of probe from not cut off too early by the exonuclease for being not intended to introduce in reaction system.Protection
Sequence with the complementary pairings such as sequence to be measured, primer, not can be simple repeated sequence, such as multiple A, T, C, G.
With reference to the accompanying drawing, testing principle of the invention is further illustrated.
By taking EGFR catastrophe point S768I as an example, the wild type of the point is G, saltant type T, during PCR, uses 1 first
To primer, ARMS primer (the last one base of primer is T) and downstream primer containing 1 for catastrophe point.The primer is quick
It is specific under the action of enzyme to amplify mutational site T.Wild type site G is not amplified, as shown in Figure 1a；
After specific amplification occurs, under the guidance of ARMS primer, quick Taq enzyme is along DNA profiling, to 5 ' -3 ' sides
Region is mediated to the sequence for moving and hydrolyze intermediary's connection primer, and the probes complementary region of intermediary's connection primer is then released,
As shown in Figure 1 b；
The probes complementary region released is based on base pair complementarity principle, with complementary, the In of universal fluorescent probe generation
In the presence of quenching group, the fluorescence of fluorophor is quenched, as illustrated in figure 1 c；
Quick Taq enzyme connects the probes complementary region of primer by intermediary, mobile to 5 ' -3 ' directions, hydrolyzes quenching group,
To which quenching group is far from fluorophor, so that PCR reaction produces fluorescence, as shown in Figure 1 d.
Therefore, as long as the ARMS primer in multiple a mutational sites, downstream primer connect primer, 1 group of universal fluorescent with intermediary
Complementary probe is quenched with general in probe, can be placed in a PCR reaction tube, it can detects prominent with the presence or absence of point in sample
Become, significantly improves the detection flux of ARMS method.
The sequence of ARMS primer used in EGFR multipoint mutation single tube quick detection kit is as follows:
ARMS primer 1:TTCAAAAAGATCAAAGTGCTGA (SEQ ID NO:1)
ARMS primer 2: TTCAAAAAGATCAAAGTGCTGT (SEQ ID NO:2)
ARMS primer 3:CAAAAAGATCAAAGTGCTGGC (SEQ ID NO:3)
ARMS primer 4:TTCCCGTCGCTATCAAAACATC (SEQ ID NO:4)
ARMS primer 5:TTCCCGTCGCTATCAAAATATC (SEQ ID NO:5)
ARMS primer 6:TTCCCGTCGCTATCAAGACATC (SEQ ID NO:6)
ARMS primer 7:TCCCGTCGCTATCAAGTCT (SEQ ID NO:7)
ARMS primer 8:CCGTCGCTATCAAGGTACA (SEQ ID NO:8)
ARMS primer 9:CGTCGCTATCAAGGCATCTC (SEQ ID NO:9)
ARMS primer 10:TCCCGTCGCTATCAAGTCT (SEQ ID NO:10)
ARMS primer 11:CCGTCGCTATCAAGGAGCCAA (SEQ ID NO:11)
ARMS primer 12:CCGTCGCTATCAAGGAGCAAT (SEQ ID NO:12)
ARMS primer 13:CCGTCGCTATCAAGGATCC (SEQ ID NO:13)
ARMS primer 14:CCGTCGCTATCAAGGAAGC (SEQ ID NO:14)
ARMS primer 15:CCGTCGCTATCAAGGAACCAA (SEQ ID NO:15)
ARMS primer 16:CCGTCGCTATCAAGGAACCAT (SEQ ID NO:16)
ARMS primer 17:CGTCGCTATCAAGGAATCTC (SEQ ID NO:17)
ARMS primer 18:CCGTCGCTATCAAGGAACCG (SEQ ID NO:18)
ARMS primer 19:CCGTCGCTATCAAGGAACAG (SEQ ID NO:19)
ARMS primer 2 0:GTCGCTATCAAGGAATCATC (SEQ ID NO:20)
ARMS primer 2 1:CGTCGCTATCAAGGAATCTC (SEQ ID NO:21)
ARMS primer 2 2:GTCGCTATCAAGGAATTCGA (SEQ ID NO:22)
ARMS primer 2 3:AGCCTACGTGATGGCCAT (SEQ ID NO:23)
ARMS primer 2 4:CCAGCGTGGCCAGCGT (SEQ ID NO:24)
ARMS primer 2 5:CCAGCGTGGACGGTAAC (SEQ ID NO:25)
ARMS primer 2 6:GACAATTCCCACCACGTGT (SEQ ID NO:26)
ARMS primer 2 7:CACCGTGCAGCTCATCAT (SEQ ID NO:27)
ARMS primer 2 8:AGATCACAGACTTTGGGCG (SEQ ID NO:28)
ARMS primer 2 9:GTTGGGCTGGCCAAACA；(SEQ ID NO:29)
The corresponding downstream primer of ARMS primer 1 and 2 is Y1:AGACCATGAGAGGCCCTG (SEQ ID NO:30)；
The corresponding downstream primer of ARMS primer 3 is Y2:AGATGATGGAAATATACAGCTTGC (SEQ ID NO:31)；
The corresponding downstream primer of ARMS primer 4~22 is Y3:TGTGGAGATGAGCAGGGT (SEQ ID NO:32)；
The corresponding downstream primer of ARMS primer 2 3~27 is Y4:TTGTCTTTGTGTTCCCGGA (SEQ ID NO:33)；
The corresponding downstream primer of ARMS primer 28 and 29 is Y5:GGCTGACCTAAAGCCACC (SEQ ID NO:34)；
The corresponding intermediary's primer of ARMS primer 1~3 is Z1:ACCTCGTTCTGGGCTCTACTCCGGTGCGTTCGGCACG
GTGTATA (SEQ ID NO:35)；
The corresponding intermediary's primer of ARMS primer 4~22 is Z2:ACCTCGTTCTGGGCTCTACTCCATGGCTCTGAACCT
CA (SEQ ID NO:36)；
The corresponding intermediary's primer of ARMS primer 2 3~27 is Z3:ACCTCGTTCTGGGCTCTACCTGCCTCCTGGACTATG
(SEQ ID NO:37)；
The corresponding intermediary's primer of ARMS primer 28 and 29 is Z4:ACCTCGTTCTGGGCTCTACCCATGCAGAAGGAGGC
AA (SEQ ID NO:38).
The sequence of special universal fluorescent probe are as follows:TC(dT-FAM)GCGGTGTTGGTGTAGAGCCCAGAACGAGGTTTTTT
TTTTTTTTTTTT (SEQ ID NO:39), it is corresponding that sequence is quenched are as follows: 5 '-BHQ-CCGCAGA-3 '.
Embodiment 1
EGFR multipoint mutation single tube quick detection kit, principal component are as follows:
DNA extracting solution:
50mM NaOH, 10mMTris-HCl (PH8.0), 1%NP-40,6%Chelex, 0.1mM EDTA (PH8.0) group
At
The quick Taq enzyme system of thermal starting:
The dNTPs (25mM) of the thermal starting Taq, 0.5ul of 3U
Primer:
The ARMS primer and corresponding downstream primer that EGFR multipoint mutation single tube quick detection kit uses are as follows:
Intermediary connects primer:
Universal fluorescent probe pair:
Universal fluorescent probe: 5 '-TC (dT-FAM) GCGGTGTTGGTGTAGAGCCCAGAACGAGGTTTTTTTTTTTTT
TTTTT-3’
General quenching probes: 5 '-BHQ-CCGCAGA-3 '.
Application method:
DNA is extracted:
The FFPE histotomy for taking 2 10 μm, is put into 1.5ml centrifuge tube after scraping down, and adds 120 μ l's
DNA extracting solution, 100 DEG C are boiled after ten minutes, and centrifuge tube 12000rpm is centrifuged 5 minutes, takes the supernatant of 10 μ l to be added to PCR anti-
Ying Guanzhong；
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 it is general be quenched each 1 μm of complementary probe, 28 mutational sites
ARMS primer, downstream primer and intermediary connect each 1 μm of primer.After 95 degree of 5min, 95 DEG C 5 seconds, 58 DEG C 31 seconds, recurring number is 40 to follow
Ring.
, such as there is point mutation, then fluorescence signal can increase, as shown in Figure 2 in the change in fluorescence for monitoring reaction system.
<110>Guangzhou two kinds of substance synthesis into another Technology Co., Ltd.
<120>a kind of multipoint mutation single tube rapid detection method and kit
<130>
<140> CN2014104295064
<141> 2014-08-28
<160> 39
<170> PatentIn version 3.5
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Claims (1)
1. multipoint mutation single tube rapid detection method, includes the following steps:
1) design connects primer, universal fluorescent probe for the ARMS primer of different point mutation and corresponding downstream primer, intermediary
And its quenching probes；Wherein, intermediary connection primer be made of universal sequence part and distinguished sequence part, universal sequence part with
Universal fluorescent probe portion sequence is complementary, the partial sequence complementarity of distinguished sequence part and point mutation downstream；
2) ARMS primer and corresponding downstream primer, intermediary's connection primer, universal fluorescent probe and its quenching probes and heat are opened
Move quick Taq enzyme system mixing, amplification；
3) change in fluorescence of reaction system is detected, it is determined whether there are point mutation；
The method is not used in the diagnosis of disease；
It is connected on universal fluorescent probe and connects the nucleotide of quenching group on the nucleotide and quenching probes of fluorophor in complementation
Spacing after pairing is no more than 4 nucleotide；
It is 15~30bp that intermediary, which connects in primer with the nucleic acid quantity of the partial sequence complementarity in mutational site downstream,；
It is 15~45bp that intermediary, which connects the nucleic acid quantity matched in primer with universal fluorescent probe portion complementary nucleic acid,；
One end of universal fluorescent probe is connected with protection nucleic acid sequence.
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| CN201410429506.4A CN104293913B (en) | 2014-08-28 | 2014-08-28 | Multipoint mutation single tube method for quick and test kit |
| CN201610532349.9A CN106148521B (en) | 2014-08-28 | 2014-08-28 | A kind of multipoint mutation single tube rapid detection method and kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104830981A (en) * | 2015-04-29 | 2015-08-12 | 广州和实生物技术有限公司 | KRAS gene multipoint mutation single tube rapid detection method and kit |
| CN105063177A (en) * | 2015-05-14 | 2015-11-18 | 广州和实生物技术有限公司 | BRAF gene multi-point mutation single tube rapid detection method and BRAF gene multi-point mutation single tube rapid detection kit |
| CN105039533A (en) * | 2015-07-08 | 2015-11-11 | 广州和实生物技术有限公司 | PIK3CA gene multipoint mutation single tube fast detection method and kit |
| CN105039534A (en) * | 2015-07-08 | 2015-11-11 | 广州和实生物技术有限公司 | C-KIT gene multipoint mutation single tube fast detection method and kit |
| CN105087792A (en) * | 2015-08-13 | 2015-11-25 | 广州和实生物技术有限公司 | Rapid single-tube detection method and rapid single-tube detection kit for multipoint mutation of PDGFRA (platelet-derived growth factor receptor alpha) gene |
| CN105087793A (en) * | 2015-08-13 | 2015-11-25 | 广州和实生物技术有限公司 | Rapid single-tube detection method and rapid single-tube detection kit for multipoint mutation of NRAS gene |
| CN105861706A (en) * | 2016-05-18 | 2016-08-17 | 健路生物科技（苏州）有限公司 | Universal probe for real-time fluorescent PCR and detection method and application of universal probe |
| CN106755489A (en) * | 2017-01-22 | 2017-05-31 | 杭州金溪生物技术有限公司 | High sensitivity detection method of gene mutation and used kit |
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| CN104293913B (en) | 2016-11-30 |
| CN106148521A (en) | 2016-11-23 |
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