CN105063177A - BRAF gene multi-point mutation single tube rapid detection method and BRAF gene multi-point mutation single tube rapid detection kit - Google Patents
BRAF gene multi-point mutation single tube rapid detection method and BRAF gene multi-point mutation single tube rapid detection kit Download PDFInfo
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- CN105063177A CN105063177A CN201510245213.5A CN201510245213A CN105063177A CN 105063177 A CN105063177 A CN 105063177A CN 201510245213 A CN201510245213 A CN 201510245213A CN 105063177 A CN105063177 A CN 105063177A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The present invention discloses a multi-point mutation single tube rapid detection method and a multi-point mutation single tube rapid detection kit. The multi-point mutation single tube rapid detection method comprises: 1) designing ARMS primers, corresponding downstream primers, intermediate connection primers, universal fluorescent probes and quenching probes; 2) mixing the ARMS primers, the corresponding downstream primers, the intermediate connection primers, the universal fluorescent probes, the quenching probes and a hot start rapid Taq enzyme system, and amplifying; and 3) detecting the fluorescence change of the reaction system, and determining whether the point mutation exists. According to the present invention, the operation is simple, and the presence of the multi-point mutation can be subjected to qualitative detection; the design difficulty of the primers used in the detection method is low so as to substantially reduce the primer synthesis cost; the limitation of the existing ARMS technology is overcome, and the detection flux is substantially improved; and the one-tube reaction replaces the previous multi-tube detection so as to save the manpower and the resources.
Description
Technical field
The present invention relates to a kind of mutation detection kit, particularly a kind of BRAF multipoint mutation single tube quick detection kit.
Background technology
Point mutation and many tumours have close relationship, there are some researches show, the point mutation in some group site can make the incidence of tumour significantly improve.For the carcinogenic autoploid B1(BRAF of Murine Sarcoma viral), BRAF gene coding serine/threonine specificity kinase, it is the most key incitant of RAS/RAF/MEK/ERK/MAPK signal path, participate in various biological event in regulating cell, as Growth of Cells, differentiation and apoptosis etc.In multiple human malignancies, as malignant melanoma, colorectal cancer, lung cancer, thyroid carcinoma, liver cancer and carcinoma of the pancreas etc. all exist the BRAF sudden change of different ratios.
In colorectal cancer patients, BRAF gene mutation rate is 15%, National Cancer complex therapy alliance " colorectal cancer clinical practice guideline " (V3.2011) advises, when using the targeted drug such as Erbitux or Victibix, tumor tissues KRAS gene appearance must be detected, if KRAS is without sudden change, should consider to detect BRAF gene state.In the treatment of melanoma patients, BRAF gene mutation rate in primary melanoma is 80%, in metastasis melanin tumor, mutation rate is 68%, mutation rate in benign nevus is up to 82%.The novel targeted medicine Vemurafenib researched and developed for BRAF gene mutation is evident in efficacy for BRAF gene mutation positive patient.
Large quantity research shows the driven nature sudden change that BRAF gene mutation occurs as papillary thyroid carcinoma (PTC) to have become an important indicator of papillary thyroid carcinoma (PTC) Fine-needle Aspiration Tissues specimens in diagnosis and state of an illness prognosis.
Mutant cell is often mixed in together with wild-type cell, therefore extracted DNA is often with a large amount of wild-type DNA, need higher specificity so detect somatic mutation, and now widely used direct sequencing detectivity is limited, can not meet clinical requirement completely.
Because the catastrophe point of BRAF gene medicaments insensitive is more, fluorescence PCR detection reagent kit on the market generally uses single gene mutation to detect, and analytic process is loaded down with trivial details and there is no need.Whole testing process is comparatively slow simultaneously, wastes a large amount of time and resource.
Be necessary the detection kit developing a kind of BRAF multipoint mutation more rapidly and efficiently.
Summary of the invention
The object of the present invention is to provide a kind of BRAF 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.
A kind of BRAF multipoint mutation single tube quick detection kit, comprise DNA extraction liquid, quick Taq enzyme system and BRAF abrupt climatic change primer, it is characterized in that: described BRAF abrupt climatic change 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 in downstream, BRAF mutational site, and the other end sequence and universal fluorescent probe portion complementary nucleic acid match.
Quick Taq enzyme system consist of dNTPS(U), UNG enzyme, fast Taq enzyme.
Preferably, the intermediary used in above-mentioned BRAF multipoint mutation single tube quick detection kit connects in primer and is no less than 5bp with the nucleic acid quantity of the partial sequence complementarity in downstream, BRAF mutational site, and especially, complementary nucleic acid quantity is 15 ~ 30bp.
Preferably, the intermediary used in above-mentioned BRAF 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 5bp, and especially, complementary nucleic acid quantity is 15 ~ 45bp.
Especially, the sequence of the ARMS primer used in above-mentioned BRAF multipoint mutation single tube quick detection kit is as follows:
V600E：AGGTGATTTTGGTCTAGCTACAGA（SEQIDNO：1）
V600Ec：AGGTGATTTTGGTCTAGCTACAGAA（SEQIDNO：2）
V600D：AGGTGATTTTGGTCTAGCTACAGAT（SEQIDNO：3）
V600K：AGGTGATTTTGGTCTAGCTACAAA（SEQIDNO：4）
V600R：ATAGGTGATTTTGGTCTAGCTACAAG（SEQIDNO：5）。
Especially, the sequence of the universal fluorescent probe used in above-mentioned BRAF multipoint mutation single tube quick detection kit is as follows: 5 '-TG (dT-FAM) CTGATCTACGTATCCACAGTGTTGACCGTGATTTTTTTTTTTTTTT-3 ' (SEQIDNO:6), and corresponding quenching probes sequence is: 5 '-BHQ-CCGCAGA-3 '.
The invention has the beneficial effects as follows:
1) of the present invention 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.
2) detection method of the present invention overcomes the limitation of existing ARMS technology, substantially increases it and detects flux.
3) detection method of the present invention achieves the simplification of multitube detection reaction, and the multitube before 1 tube reaction instead of detects.Use manpower and material resources sparingly.
4) 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.
With BRAF catastrophe point V600E for example, the wild-type of this point is T, 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, and wild type site T is not amplified.As shown in Figure 1;
After specific amplification occurs, under the guiding of ARMS primer, quick Taq enzyme is along DNA profiling to 5 '-3 ' direction moves and is hydrolyzed intermediary and connect the sequence mediates region of primer, and 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 site, 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
BRAF multipoint mutation single tube quick detection kit, principal constituent is as follows:
1) DNA extraction liquid:
50mMNaOH, 10mMTris-HCl(PH8.0), 1%NP-40,6%Chelex, 0.1mMEDTA(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 BRAFARMS primer and correspondence:
Intermediary connects primer:
4) universal fluorescent probe is to as follows:
Universal fluorescent probe: 5 '
-TG (dT-FAM) CTGAtCTACGTATCCACAGTGTTGACCGTGATTTTTTTTTTTTTTT-3 '
General quenching probes: 5 '
bHQ-TCAGACA-3 '
Use BRAF 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 10mMTris-HCL, 50mMKCL, 0.5U, 0.2mMdNTPS(U), 3mMMgCl2.Universal fluorescent probe and each 1 μm of general cancellation complementary probe, the ARMS primer in 5 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;
3) monitor the change in fluorescence of 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>BRAF gene multipoint mutation single tube method for quick and test kit
<130>
<160>8
<170>PatentInversion3.5
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The artificial primer of <213>
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ataggtgattttggtctagctacaag26
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Claims (9)
1. multipoint mutation single tube method for quick, comprises the steps:
The 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;
By the downstream primer of ARMS primer and correspondence, intermediary connects primer and universal fluorescent probe mixes with quick Taq enzyme system, increases;
The change in fluorescence of detection reaction system, determines whether there is point mutation.
2. a BRAF multipoint mutation single tube quick detection kit, comprise DNA extraction liquid, quick Taq enzyme system and BRAF abrupt climatic change primer, it is characterized in that: described BRAF abrupt climatic change 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 in downstream, BRAF mutational site, and the other end sequence and universal fluorescent probe portion complementary nucleic acid match.
3. BRAF 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. BRAF multipoint mutation single tube quick detection kit according to claim 2, is characterized in that: intermediary connects in primer and is no less than 5bp with the nucleic acid quantity of the partial sequence complementarity in downstream, BRAF mutational site.
5. BRAF 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 in downstream, BRAF mutational site.
6. BRAF 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 5bp.
7. BRAF 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 BRAF 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. BRAF multipoint mutation single tube quick detection kit according to claim 8, is characterized in that: the sequence of universal fluorescent probe is: 5 '
-TG (dT-FAM) CTGAtCTACGTATCCACAGTGTTGACCGTGATTTTTTTTTTTTTTT-3 ', corresponding cancellation sequence is: 5 '-BHQ-CCGCAGA-3 '.
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| CN107400716A (en) * | 2017-08-24 | 2017-11-28 | 上海生物芯片有限公司 | PCR detection method, primer, probe and the kit of people's BRAF gene V600E mutation |
| CN109161594A (en) * | 2018-08-17 | 2019-01-08 | 中山大学达安基因股份有限公司 | A kind of method and its kit detecting BRAF gene mutation |
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