CN108048569A - The primer and probe of BRAF gene mutation detection and application - Google Patents

The primer and probe of BRAF gene mutation detection and application Download PDF

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CN108048569A
CN108048569A CN201711469163.4A CN201711469163A CN108048569A CN 108048569 A CN108048569 A CN 108048569A CN 201711469163 A CN201711469163 A CN 201711469163A CN 108048569 A CN108048569 A CN 108048569A
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primer
probe
gene mutation
seq
kit
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廖军华
郑群艳
叶滔
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Guangzhou Reputation Biotechnology Co Ltd
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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
    • 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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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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Abstract

Primer and probe and application the invention discloses BRAF gene mutation detection, belong to technical field of gene detection, the primer includes the universal primer for expanding wild type gene and mutated genes simultaneously, and only expand the mutant primer of mutated genes, the nucleotide sequence of the mutant primer is as shown in SEQ ID NO.1 and SEQ ID NO.2, the nucleotide sequence of the universal primer is as shown in SEQ ID NO.3 and SEQ ID NO.4, and the nucleotide sequence of the probe is as shown in SEQ ID NO.5.The primer of the present invention and spy are applied in fluorescence quantitative PCR detection, it turns out that easy to operate, low-cost and accuracy is high.

Description

The primer and probe of BRAF gene mutation detection and application
Technical field
The primer and probe that is detected the invention belongs to technical field of gene detection more particularly to BRAF gene mutation and should With.
Background technology
BRAF gene is a member in RAF gene families, positioned at No. 7 chromosome it is long-armed on, coding one contains 766 The protein of amino acid residue.Braf albumen is a kind of serine threonine protein kinase, participates in a variety of lifes in regulating cell Object activity transduces signal to MEK1/2 from ras in MAPK accesses.BRAF gene mutation causes the protein kinase activity Variation is generated, cell is caused abnormal multiplication and differentiation occur, ultimately forms tumour.
BRAF gene mutation, wherein colorectal cancer, melanoma and thyroid gland all have occurred in nearly 8% human tumor Papilloma occurs more.It is at most that codon T becomes A in the 1799th amino acids in BRAF gene mutation (BRAFV600E), accounting 92%.There are two class BRAF inhibitor at present, one kind is the RAF kinase inhibitors of wide spectrum, suitable for more Type, but specific aim is not strong；Another kind of is BRAFV600E targeted inhibition agent, there is very high inhibitory action to BRAFV600E. Advanced melanoma patient outcome caused by targeted inhibition agent Wei Luofeini treats this kind mutation is notable, but drug resistance makes this kind The using effect of drug is significantly limited, resistance mechanism, new drug development and how to avoid or reduce drug resistance be urgently to solve at present Certainly the problem of.
The technical method of genetic test at present has Sanger sequencings, RNase patterning method, denaturing gradient gel electrophoresis (Denatured Gradient Gel Electrophoresi, DGGE), heteroduplex analysis method (HA), chemical cleavage mispairing Method (CCM), Sorpions-ARMS (Scorpions, scorpion shape probe；Amplification Refractory Mutation System, amplification refractory mutation system), DNA chip technology, TaqMan-qPCR, DHPLC (Denaturing high performance liquid chromatography)、PCR-SSCP(Single-Strand Conformation Polymorphism), PCR-RFLP (Restriction Fragment Length Polymorphism) etc., wherein Sanger Sequencing and Scorpions-ARMS apply more in clinical and scientific research.German Qiagen companies are based on Scorpions-ARMS methods Detection kit is developed, which is high sensitivity, easy to operate and detection is quick, but its cost is excessively high.Sanger PCR sequencing PCR is the goldstandard of detection in Gene Mutation and gene diversity detection, and platform and technology are all more mature, has both detection knot Fruit is accurate, comprehensive, and testing cost is cheap and can detect unknown mutation；But Sanger PCR sequencing PCRs are limited to mutator content will Reach more than 10%, and need post-processing, it is easily contaminated.
Although conventional fluorescent quantitative PCR method is widely used, but accuracy has much room for improvement.Therefore, it is necessary to design one Easy to operate, the low-cost and high accuracy fluorescence quantitative PCR detection of kind.
The content of the invention
Present invention aims to overcome that the shortcomings of the prior art, and a kind of detection mankind's BRAF gene mutation is provided Primer and probe and application, the primer and probe be applied to fluorescence quantitative PCR detection in, it turns out that it is easy to operate, expense is low Honest and clean and accuracy is high.
To achieve the above object, the technical solution taken of the present invention is：The primer of BRAF gene mutation detection, including simultaneously Expand the universal primer of wild type gene and mutated genes and only expand the mutant primer of mutated genes, the mutation is drawn The nucleotide sequence of object is as shown in SEQ ID NO.1 and SEQ ID NO.2, the nucleotide sequence such as SEQ ID of the universal primer Shown in NO.3 and SEQ ID NO.4, the BRAF gene mutation is c.1799T ＞ A.
In addition, the answering in the kit for detecting BRAF gene mutation is prepared the present invention also provides the primer With.
In addition, the present invention also provides the probe of BRAF gene mutation detection, the nucleotide sequence such as SEQ ID of the probe Shown in NO.5, the BRAF gene mutation is c.1799T ＞ A.
As the improvement of above-mentioned technical proposal, the both ends difference mark fluorescent reporter group and fluorescent quenching base of the probe Group, the fluorescent reporter group are selected from FAM, VIC, HEX, Cy5And Cy3In one kind, the quenching group be selected from BQH1、 MGB, TAMARA and BHQ2In one kind.
In addition, the answering in the kit for detecting BRAF gene mutation is prepared the present invention also provides the probe With.
In addition, the present invention also provides a kind of kits, it includes the primer and/or the probes.
As the improvement of above-mentioned technical proposal, the kit also includes RNA extracts reagents, cDNA synthetic agents and PCR Reaction reagent.
As being further improved for above-mentioned technical proposal, the PCR reaction reagents include PCR buffer solutions, dNTPs and DNA Polymerase.
As the further improvement of above-mentioned technical proposal, when PCR amplification system is 20 μ L, amplification system contains following Component：10 μ L of PCR mixed liquors, 1 μ L of cDNA templates, 7.5 μ L of every 0.5 μ L of primer, 0.5 μ L of probe and nuclease free pure water；
Amplification program is：95 DEG C of pre- change 10min；94 DEG C of denaturation 15s, 60 DEG C extend 1min, cycle 40 times；50℃2min.
The beneficial effects of the present invention are：The present invention provide it is a kind of detect mankind's BRAF gene mutation primer and probe and Using the mutant primer and probe that the present invention designs are when detection is without BRAFV600E sudden change samples, the CT values of amplification curve Greatly, the high specificity of mutant primer；Universal primer to avoid false negative result, can make easy to operate, the expense of quantitative fluorescent PCR Cheap and accuracy is high.
Description of the drawings
Fig. 1 is that there are the positive test symbol figures of BRAF gene mutation in sample；
Fig. 2 is the negative result figure that BRAF gene mutation is not present in sample.
Specific embodiment
For the object, technical solutions and advantages of the present invention are better described, below in conjunction with specific embodiment, attached drawing to this Invention is described further.
Embodiment 1
The present embodiment provides a pair of for detecting the primer of BRAF gene mutation, including expanding wild type gene and mutation simultaneously The universal primer of type gene and the mutant primer for only expanding mutated genes, the nucleotides sequence of mutant primer F are classified as：5’- TCATAATGCTTGCTCTGATAGG-3 ', the nucleotides sequence of mutant primer R are classified as：3’-CCA AAAATTTAATCAGTGGA- 5’；The nucleotides sequence of universal primer F is classified as：5 '-TAGGTGATTTTGGTCTAGCTACTT-3 ', the nucleotides sequence of universal primer R It is classified as：5’-TAGTTGAGACCTTCAATGACTTTCTAGT-3’.
Embodiment 2
The present embodiment provides a pair of for detecting the probe of BRAF gene mutation, the nucleotides sequence of probe is classified as：5’-FAM- TGGAGTGGGTCCCATCAGTTTG-BQH1-3’。
Embodiment 3
The present embodiment provides a pair of for detecting the kit of BRAF gene mutation, the kit includes 1 institute of embodiment The probe described in primer and embodiment 2 stated.
Embodiment 4
The present embodiment provides a kind of for detecting the kit of BRAF gene mutation, with embodiment 3 difference lies in：This Kit further includes the kit also comprising RNA extracts reagents, cDNA synthetic agents, PCR reaction reagents in embodiment, described PCR reaction reagents include PCR buffer solutions, dNTPs and archaeal dna polymerase.
When PCR amplification system is 20 μ L, amplification system contains following components：10 μ L of PCR mixed liquors, 1 μ L of cDNA templates, 7.5 μ L of every 0.5 μ L of primer, 0.5 μ L of probe and nuclease free pure water；
Amplification program is：95 DEG C of pre- change 10min；94 DEG C of denaturation 15s, 60 DEG C extend 1min, cycle 40 times；50℃2min.
Fluorescent quantitative PCR detection method
1. Total RNAs extraction
Need the reagent prepared：Chloroform, isopropanol, 75% ethyl alcohol (being prepared with RNase-free water), RNase-free Water.
1) homogenized (Homogenization)
It directly takes fresh blood, adds in 3 times of volume erythrocyte cracked liquids, be placed at room temperature for 10min after mixing, 10, 000rpm centrifuges 1min；It thoroughly inhales and abandons supernatant, collect leukocyte cell pellet, the leukocyte cell pellet of every 100~200 μ L blood collections adds Enter 1mL TRIzol；
2) it is layered (Phase Separation)
After sample adds in TRIzol, 5min is placed at room temperature for, sample is made fully to crack and (is such as operated without next step, sample - 80 DEG C long-term preserve can be put into)；Optional step：4 DEG C of 12,000rpm centrifuge 10min, take supernatant；If containing more in sample Albumen, fat, polysaccharide or muscle, plant tuberal part grade, and can centrifuge removal；When handling adipose tissue sample, upper strata is a large amount of Grease should remove；Clear homogenate solution is taken to carry out next step operation, 200 μ L chloroforms are added in per 1mL TRIzol, are acutely vibrated 3~5min is placed at room temperature for after mixing makes its natural split-phase；
3) RNA precipitate (RNA Precipitation)
4 DEG C of 12,000rpm centrifuge 10~15min, and sample can be divided into three layers：The organic phase of yellow, interlayer and colourless Water phase；Water phase (can usually draw 550 μ l) mainly in water phase, is transferred to new pipe by RNA；It is added in supernatant isometric ice-cold Isopropanol, be placed at room temperature for 10~20min；4 DEG C of 12,000rpm centrifuge 10min, abandon supernatant, RNA precipitate is in tube bottom；
4) RNA rinses (RNA Wash)
75% ethyl alcohol of 1mL (being prepared with RNase-free water) is added in RNA precipitate, mildly vibrates centrifuge tube, it is heavy to suspend It forms sediment；75% ethyl alcohol of 1mL is added in per 1mL TRIzol, 4 DEG C 5,000~8,000rpm centrifuges 1~2min, abandons supernatant, is placed at room temperature for 1~2min dries precipitation；
5) dissolve
50~100 μ L RNase-free water are added in RNA precipitate, flick tube wall, fully to dissolve RNA, -80 DEG C of preservations.
2. remove genome
Using the DNase I of RNase-free, by following system configurations reaction solution, 37 DEG C of digestion 30min, 65 DEG C of inactivations 10min；System includes following components：60 μ L, DNase I of RNA 20 μ L, 10 × buffer 20 μ L, H2O 100μL；
Then operate according to the following steps：
1) add in isometric phenol, 10,000rpm after the mixing that turns upside down, centrifuge 5min；
2) supernatant is taken, adds in isometric chloroform, turn upside down mixing, and 10,000rpm, centrifuge 10min；
3) supernatant is taken, adds in isometric isopropanol, gently abundant mixing, -20 DEG C of standing 15min；
4) 4 DEG C of 10,000g centrifugation 10min, remove supernatant；
5) washed twice with 75% ethyl alcohol, super-clean bench air-dries；
6) 10 μ L DEPC water dissolutions are added in.
3.RNA concentration, purity and integrity detection
Purity detecting：RNA sample is taken moderately to dilute, OD values, OD260/ are measured on micro-spectrophotometer k2800 The ratio of OD280 is more than 1.8, illustrates that the RNA prepared is purer, no protein contamination.
4. reverse transcription
1) RNA, primer mixture, 10 μ L of total amount are added in PCR pipe, system includes following components：RNA 15 μ g, Olig (dT) 18 0.5 μ L, Random primer, 0.5 μ L, RNase free ddH2O complements to 10 μ L；
2) rapidly in cooled on ice 2min after 70 DEG C of heat preservation 10min；
3) following reagent is added in the PCR pipe：12 μ L, 10mM dNTP Mixture of RNA/ primers denaturing soln, 0.5 μ 0.25 μ L, 5 × M-MLV buffer of L, RNase inhibitor (40U), 4 μ L, RTase M-MLV, 0.5 μ L, RNase free ddH2O complements to 20 μ L；
4) by above-mentioned 20 μ L reaction solutions, 42 DEG C of heat preservation 60min；72 DEG C of heat preservation 15min；- 20 DEG C save backup.
5. design of primers
Primer is designed at the extron both ends containing mutational site using Primer Premier 5.0, is avoided with primer Dimer and the sequence of stem ring mispairing are amplified the sequence length come using it and are no more than 400bp, and the annealing of the primer Temperature is basically identical；Under institute's amplimer of the present invention and the connection sequence of primer such as table 1.
Table 1
6. fragment amplification
20 μ L amplification systems are as shown in table 2；
Table 2
Reagent Usage amount
PCR mixed liquors 10μL
Primers F (10 μM) 0.5μL
Primer R (10 μM) 0.5μL
Mutant probe 0.5μL
CDNA templates 1μL
Nuclease free pure water 7.5μL
Amplification program is：95 DEG C of pre- change 10min；94 DEG C of denaturation 15s, 60 DEG C extend 1min, cycle 40 times；50℃2min.
7. result
As shown in Figure 1, showing two positive amplification curves in figure, illustrate that test sample contains BRAF gene mutation；Such as Fig. 2 It is shown, a positive amplification curve is shown in figure, illustrates that test sample does not contain BRAF gene mutation.
It can be seen that the present invention mutant primer and probe detection without BRAFV600E sudden change samples when, amplification curve CT values it is big, the high specificity of mutant primer；Universal primer to avoid false negative result, can make the accuracy of quantitative fluorescent PCR It is high.
Finally, it should be noted that above example to illustrate technical scheme rather than to the present invention protect The limitation of scope, although being explained in detail with reference to preferred embodiment to the present invention, those of ordinary skill in the art should manage Solution, can modify to technical scheme or replace on an equal basis, without departing from technical solution of the present invention essence and Scope.
SEQUENCE LISTING
<110>Guangzhou Yu Jia bio tech ltd
<120>The primer and probe of BRAF gene mutation detection and application
<130> 2017
<160> 5
<170> PatentIn version 3.3
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<211> 22
<212> DNA
<213>It is artificial synthesized
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tcataatgct tgctctgata gg 22
<210> 2
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 2
ccaaaaattt aatcagtgga 20
<210> 3
<211> 24
<212> DNA
<213>It is artificial synthesized
<400> 3
taggtgattt tggtctagct actt 24
<210> 4
<211> 28
<212> DNA
<213>It is artificial synthesized
<400> 4
tagttgagac cttcaatgac tttctagt 28
<210> 5
<211> 22
<212> DNA
<213>It is artificial synthesized
<400> 5
tggagtgggt cccatcagtt tg 22

Claims (9)

  1. The primer of 1.BRAF detection in Gene Mutation, which is characterized in that including expanding the logical of wild type gene and mutated genes simultaneously With primer and only expand the mutant primer of mutated genes, the nucleotide sequence such as SEQ ID NO.1 of the mutant primer and Shown in SEQ ID NO.2, the nucleotide sequence of the universal primer is described as shown in SEQ ID NO.3 and SEQ ID NO.4 BRAF gene mutation is c.1799T ＞ A.
  2. 2. application of the primer as described in claim 1 in the kit for detecting BRAF gene mutation is prepared.
  3. The probe of 3.BRAF detection in Gene Mutation, which is characterized in that the nucleotide sequence of the probe such as SEQ ID NO.5 institutes Show, the BRAF gene mutation is c.1799T ＞ A.
  4. 4. probe as claimed in claim 3, which is characterized in that the both ends difference mark fluorescent reporter group and glimmering of the probe Optical quenching group, the fluorescent reporter group are selected from FAM, VIC, HEX, Cy5And Cy3In one kind, the quenching group is selected from In BQH1, MGB, TAMARA and BHQ2In one kind.
  5. 5. application of the probe in the kit for detecting BRAF gene mutation is prepared as described in claim 3 or 4.
  6. 6. a kind of kit, which is characterized in that include the spy described in primer as described in claim 1 and/or claim 3 Pin.
  7. 7. kit as claimed in claim 6, which is characterized in that the kit is also synthesized comprising RNA extracts reagents, cDNA Reagent and PCR reaction reagents.
  8. 8. kit as claimed in claim 7, which is characterized in that the PCR reaction reagents include PCR buffer solutions, dNTPs and Archaeal dna polymerase.
  9. 9. kit as claimed in claim 8, which is characterized in that when PCR amplification system be 20 μ L when, amplification system contain with Lower component：10 μ L of PCR mixed liquors, 1 μ L of cDNA templates, 7.5 μ L of every 0.5 μ L of primer, 0.5 μ L of probe and nuclease free pure water；
    Amplification program is：95 DEG C of pre- change 10min；94 DEG C of denaturation 15s, 60 DEG C extend 1min, cycle 40 times；50℃2min.
CN201711469163.4A 2017-12-27 2017-12-27 The primer and probe of BRAF gene mutation detection and application Pending CN108048569A (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070020657A1 (en) * 2005-05-20 2007-01-25 Grebe Stefan K Methods for detecting circulating tumor cells
CN102154480A (en) * 2011-01-28 2011-08-17 陈唯军 One-step detection method of genetic mutation and B-raf genetic point mutation, and kit thereof
CN102242207A (en) * 2011-06-29 2011-11-16 浙江大学 Primers and probes for detecting mutation of cancer gene BRAFV600E
CN104099425A (en) * 2014-08-01 2014-10-15 上海赛安生物医药科技有限公司 B-raf gene mutation detection kit
CN104846106A (en) * 2015-05-29 2015-08-19 沈阳优吉诺生物科技有限公司 Primer and kit for detecting BRAF gene V600E mutation sites, and PCR method of kit

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070020657A1 (en) * 2005-05-20 2007-01-25 Grebe Stefan K Methods for detecting circulating tumor cells
CN102154480A (en) * 2011-01-28 2011-08-17 陈唯军 One-step detection method of genetic mutation and B-raf genetic point mutation, and kit thereof
CN102242207A (en) * 2011-06-29 2011-11-16 浙江大学 Primers and probes for detecting mutation of cancer gene BRAFV600E
CN104099425A (en) * 2014-08-01 2014-10-15 上海赛安生物医药科技有限公司 B-raf gene mutation detection kit
CN104846106A (en) * 2015-05-29 2015-08-19 沈阳优吉诺生物科技有限公司 Primer and kit for detecting BRAF gene V600E mutation sites, and PCR method of kit

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
阮红等: "《基因工程原理》", 30 September 2007, 浙江大学出版社 *

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