CN104017887A - Primer pair as well as probe and kit for detecting human BRAF gene mutation - Google Patents
Primer pair as well as probe and kit for detecting human BRAF gene mutation Download PDFInfo
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- CN104017887A CN104017887A CN201410274543.2A CN201410274543A CN104017887A CN 104017887 A CN104017887 A CN 104017887A CN 201410274543 A CN201410274543 A CN 201410274543A CN 104017887 A CN104017887 A CN 104017887A
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Abstract
The invention discloses a primer pair and a probe for detecting a human BRAF gene mutation. The sequences of the primer pair and probe are shown as SEQ ID NO: 1 to SEQ ID NO: 7. The invention also discloses a kit for detecting human BRAF gene mutation and used for detecting T-A base mutation occurring on 1799-postion nucleotide of the BRAF gene exon 15 by a fluorescent PCR. The kit disclosed by the invention has the advantages of simple and quick operation, high specificity and low misdetection rate and is applicable to the auxiliary clinical diagnosis and typing of thyroid cancer, or provides a clinical reference for selecting tyrosine kinase inhibitors of melanoma and colorectal cancer and BRAF gene mutation targeted drugs.
Description
Technical field
The present invention relates to a kind of biological detection reagent, in particular, the present invention relates to a kind of mankind BRAF detection in Gene Mutation primer pair and probe and adopt the detection kit of this primer pair and probe.
Background technology
BRAF gene, the carcinogenic autoploid B1 of full name muroid sarcoma filterability toadstool (V-Raf), RAF gene family member, coding protein serine/threonine, it is the effector of oncogene RAS, also be MAPK cascade signal transduction assembly, relate to multiple physiological processs, participate in the processes such as the growth of regulating cell inner cell, differentiation and apoptosis.In multiple human malignancies, the sustained activation that BRAF transgenation causes can make the disorder of MEK/ERK signal transduction, causes cell hyperproliferation, occurs vicious transformation, suddenlys change as melanoma, colorectal cancer, thyroid carcinoma all exist the BRAF of different ratios.The BRAF transgenation of about 80-90% occurs on 1799 Nucleotide of exons 15, and T sports A, causes α-amino-isovaleric acid to be replaced (V600E) by L-glutamic acid.
BRAF genosome cell mutation is more common in papillary thyroid carcinoma, BRAF V600E sudden change is considered to that PTC is modal can heritable variation, cancer databases (COSMIC) is delivered report and is shown on its website, and 78000 examples exist and in the sample of BRAF transgenation, exceed 95% and suddenly change for V600E.The driven nature that BRAF transgenation occurs as papillary thyroid carcinoma (PTC) is suddenlyd change, and has become an important indicator of PTC Fine-needle Aspiration Tissues specimens in diagnosis and state of an illness prognosis.BRAF gene mutation rate up to 44%, has good diagnostic significance in PTC.The thyroid carcinoma patient of the BRAF positive gene mutation patients with negative prognosis mala that suddenlys change.
In melanomatous research, find, BRAF V600E sudden change has occurred in melanoma patients for approximately 50% late period, and in some researchs, its mutation rate is even higher than 60%.2011, first novel B RAF V600E target inhibitor Wei Luofeini (Vemurafenib) is by FDA approval listing, it can select to block RAF/MEK/ERK passage, suppress the melanoma cell propagation of BRAF sudden change, evident in efficacy, can obviously extend Progression free survival phase and Overall survival compared with chemicotherapy.
In colorectal cancer research, BRAF transgenation is a poor biomarker of colorectal carcinoma prognosis, and distinguishes Sporadic Colorectal Carcinoma and the syndromic molecular indexes of Lynch.In addition, may there is former resistance to EGFR targeted drug in the patient of BRAF transgenation, therefore, American National cancer complex therapy alliance (NCCN) " colorectal cancer clinical practice guideline " (2013) suggestion, in the time using the targeted drugs such as Erbitux (Cetuximab) or Victibix (Panitumumab), KRAS gene wild-type patient is further detected to BRAF gene appearance.
At present, the detection analytical technique of transgenation is mainly comprised: the methods such as order-checking, restriction fragment length polymorphism method (RFLP), single strand conformation polymorphism (SSCP), sex change high performance liquid chromatography (DHPLC), gene chip, liquid-phase chip.These methods have advantage and defect separately, still have much room for improvement and stdn.
1. direct Sequencing: Sanger sequencing, because of the variation that can read the each base of DNA, is considered to detect the gold standard method of transgenation at present.But because sensitivity low (detect sudden change sensitivity and be about 20%), while detecting heterogeneous higher clinical tumor tissue samples, false negative rate is high.Meanwhile, order-checking complex steps, length consuming time, to having relatively high expectations of equipment and operator, sense cycle is long, be limited in R&D institution more and carry out, most of hospitals all cannot be detected by complete independently, the in-vitro diagnosis product that be not easy to form normalizing operation, is applicable to clinical unit's application.
2. restriction fragment length polymorphism method (RFLP): the method cost is low, low to test set requirement, the detection limit of mutator gene is 5～10%.But labour intensity is slightly large, need technical professional, be unsuitable for high throughput testing and extensive clinical application.
The method complicated operations, high to equipment requirements such as 3.SSCP, DHPLC, gene chip, liquid-phase chip, only in R&D institution's laboratory applications, are not suitable for promoting the use of on a large scale.
Summary of the invention
In view of this, of the present invention solved technical problem is to overcome the deficiencies in the prior art, and a kind of detection primer pair and probe that detects better effects if, specificity is high, loss is low mankind BRAF transgenation is provided.
Of the present invention solved technical problem is also to provide a kind of detection kit of simple and quick, specificity is high, loss is low mankind BRAF transgenation.
In order to solve the problems of the technologies described above, on the one hand, the invention provides a kind of mankind BRAF detection in Gene Mutation primer pair and probe, detect for fluorescent PCR, the sequence of described primer pair and probe is as follows:
Described primer pair comprise for the Auele Specific Primer of containing BRAF gene mutation site to the internal control primer pair of monitoring sample DNA quality, described Auele Specific Primer is to being:
B-AR-F：GTGATTTTGGTCTAGCTACACA<SEQ?ID：1>，
B-AR-R：CTAGTAACTCAGCAGCATCTCA<SEQ?ID：2>；
Described internal control primer pair is:
B-IC-F：ATTGTTACCCAGTGGTGTGA<SEQ?ID：4>，
B-IC-R：TCGTGCAATATCTATAAGTTTGA<SEQ?ID：5>；
Described probe comprises the internal control probe for the specific probe of BRAF gene mutation site and monitoring sample DNA quality, specific probe sequence is sequence as shown in SEQ ID:3 or the complementary sequence of this sequence, and internal control probe sequence is sequence as shown in SEQ ID:6 and SEQ ID:7 or the sequence with the sequence complementation shown in SEQ ID:6 and SEQ ID:7.
Preferably, described BRAF gene mutation site is the T → A base mutation occurring on 1799 Nucleotide of BRAF gene extron 15.
Preferably, described probe is fluorescence labeling probe, and the specific probe after fluorescent mark is:
B-AR-P：6-FAM-ACCCTGGAGTGGGTCCCATCAGTTTGGG-BHQ1<SEQ?ID：3>；
Internal control probe after fluorescent mark is:
B-IC-P1：HEX-CAGCTTGTATCACCATCTCCATAT-NH2<SEQ?ID：6>
B-IC-P2：GATATGGAGATGGTGATACAAGCT-Dabcyl<SEQ?ID：7>。
On the other hand, the present invention also provides a kind of test kit of mankind BRAF detection in Gene Mutation, detects for fluorescent PCR, and described test kit comprises the primer pair and the probe that in such scheme of the present invention, disclose.
Preferably, described test kit also comprises positive control and negative control.
Preferably, described test kit comprises reaction solution, is dissolved with described primer pair and probe in reaction solution, and reaction solution also comprises PCR damping fluid, dNTP mixed solution, MgCl
2, Taq enzyme and remove DNA enzyme distilled water.
Preferred, in described reaction solution, the reaction density of each component is: described Taq enzyme 0.1-0.5U/ μ L, each primer 0.1-0.5 μ M, each probe 0.1-0.5 μ M in primer pair, PCR damping fluid 1 ×, dNTP mixed solution 0.5-1mM, MgCl
22.0-5.0mM, go DNA enzyme distilled water to add to configuration 1 × reaction system cumulative volume.
What the present invention adopted is the fluorescent PCR method that combines ARMS (Amplification refractory mutation system, amplification refractory mutation system) Auele Specific Primer and two kinds of technology of fluorescent probe.Detecting principle is with Auele Specific Primer enrichment mutator gene fragment, improves the signal of mutator gene in sample to be checked, then detects target amplicon with fluorescent probe, sensitivity and specificity that guaranteed reagent,G.R. box detects.The internal control primer, the probe that arrange with pipe, in order to monitor sample DNA quality, avoid detected result to occur false negative.According to fluorescent PCR detected result, be fluorescent PCR instrument calculate Ct value judgement sample in BRAF gene V600E mutation status, for auxiliary clinical diagnosis of thyroid cancer, somatotype, or tyrosine kinase inhibitor (EGFR-TKIs) to melanoma, colorectal cancer and BRAF transgenation targeted drug are selected clinical reference are provided.
Beneficial effect of the present invention is:
(1) adopt the detection system of specific primer, fluorescent probe and optimization, can detect the mutation status of the somatic BRAF gene of tumour patient V600E.
(2) highly sensitive, detect 50ng human gene group DNA with 25 μ L detection system, can detect contained 1% the mutant nucleotide sequence that is low to moderate.
(3) specificity is good, and DNA is without non-specific amplification signal for 50ng people's wild type gene group.
(4) detection speed is fast, and testing process only needs to complete for 90 minutes.
(5) detected result can be assisted clinical diagnosis of thyroid cancer, somatotype, or tyrosine kinase inhibitor (EGFR-TKIs) to melanoma, colorectal cancer and BRAF transgenation targeted drug are selected clinical reference is provided.
Brief description of the drawings
The PCR result figure that Fig. 1 (A)-Fig. 1 (D) is the standard substance that contain mutant plasmid under BRAF gene V600 wild-type clinical sample in embodiment 1 and 50ng people's wild type gene group DNA background, wherein:
Fig. 1 (A): the FAM channel signal of BRAF gene V600 wild-type clinical sample,
Fig. 1 (B): the VIC channel signal of BRAF gene V600 wild-type clinical sample,
Fig. 1 (C): the FAM channel signal of the standard substance that contain mutant plasmid under 50ng people's wild type gene group DNA background,
Fig. 1 (D): the VIC channel signal of the standard substance that contain mutant plasmid under 50ng people's wild type gene group DNA background.
Fig. 2 (A) and Fig. 2 (B) they are the PCR result figure of thyroid carcinoma sample in embodiment 3, wherein:
Fig. 2 (A): FAM channel signal,
Fig. 2 (B): VIC channel signal.
Fig. 3 (A) and Fig. 3 (B) they are the PCR result figure of melanoma sample in embodiment 4, wherein:
Fig. 3 (A): FAM channel signal,
Fig. 3 (B): VIC channel signal.
Fig. 4 (A) and Fig. 4 (B) they are the PCR result figure of colorectal cancer sample in embodiment 5, wherein:
Fig. 4 (A): FAM channel signal,
Fig. 4 (B): VIC channel signal.
Embodiment
The present invention is directed to the demand that in " the state-run cancer integrated network of the U.S. (NCCN) clinical practice guideline ", thyroid carcinoma, melanoma, colorectal cancer patients is detected BRAF gene V600E mutation status, the mankind BRAF gene wild-type sequence of announcing according to Cosmic data, take 1799 mutational sites (V600E) as basis as functional catastrophe point, design Auele Specific Primer and probe, and the test kit that contains Auele Specific Primer and specific probe.
Detection method of the present invention is presented below:
1. sample process and DNA extraction: detecting sample is the DNA that the fixing paraffin embedding of neutral formalin tumor tissue section extracts.The collection of tumor tissues is advisable to reject to greatest extent healthy tissues.Paraffin section thickness is 6 μ m～10 μ m, makes routine pathology section (on common slide glass) or directly several pieces tissue slicies is placed in to centrifuge tube.Recommendation QIAamp DNA FFPE Tissue Kit or extract DNA with the commercialization DNA extraction test kit of performance.With spectrophotometer, the DNA storing solution after extracting is carried out to concentration determination, should at least reach 10ng/ μ L, and OD
260nm/ OD
280nmratio should be in 1.6-2.0 scope.The working fluid that DNA is diluted to 10ng/ μ L is for subsequent use.
2. reaction system preparation and amplification:
A) preparation mixed solution (Mix), the preparation of ratio shown according to the form below.
| ? | Every reaction volume (μ L) | N reaction volume (μ L) |
| Detection reaction liquid | 20 | 20×N |
| Taq enzyme | 1 | 1×N |
B) mixed solution packing, adds 20 μ L Mix by the every hole/pipe of stoichiometric number.
C) add template, in positive control reacting hole/pipe, negative control reacting hole/pipe, sample reacting hole/pipe, add successively respectively in order 5 μ L positive controls, negative control, sample DNA (working fluid that the sample DNA suggestion that paraffin section extracts is diluted to 10ng/ μ L uses).Wherein, positive control refers to the reference standards that T → A base mutation has occurred at 1799 Nucleotide of BRAF gene extron 15, and negative control refers to wild-type reference standards.
D) in reaction system, each component final concentration is as follows:
Wherein, the component of reaction system comprises primer and the probe as shown in SEQ ID:1～SEQ ID:7, synthetic by Sangon Biotech (Shanghai) Co., Ltd..
E) real-time PCR reactions condition is as follows:
Reaction volume: 25 μ L; The denaturation stage: 95 DEG C 10 minutes, 1 circulation; The amplification stage: 95 DEG C 30 seconds, 61 DEG C 1 minute, 50 circulations; Signal collection: collect FAM and HEX (or VIC) signal in the time of 61 DEG C of amplification stages.
3. detect FAM and HEX passage fluorescent signal Ct value, according to Ct value judged result: detect FAM and HEX passage amplification curve, reaching setting threshold (Ct is less than 30) with HEX signal is to show that DNA effective content is in allowed band, FAM channel signal credible result; While reaching the threshold value of setting using FAM channel signal, needed Ct value is as yin and yang attribute criterion: Ct < 48, and pattern detection is positive; Ct >=48 or without amplification, the negative or mutant nucleotide sequence copy number of pattern detection is lower than the detection lower limit of test kit.In order to make the present invention easier to understand, will further set forth specific embodiments of the invention below.
Embodiment 1
To be DNA (company standard product) taking BRAF gene V600 wild-type clinical sample, the BRAF gene V600 wild-type cell that is mixed with mutant plasmid set forth detection performance of the present invention as example to this example.
Experiment is verified specificity of the present invention with the clinical paraffin-embedded tissue sample of BRAF gene V600 wild-type; Verify repeatability of the present invention with the company standard product of high mutation rate and low mutation rate respectively, verify sensitivity of the present invention with 1% standard substance.
Detection comprises the following steps:
1.DNA extracts
For clinical paraffin-embedded tissue sample, get thickness and be approximately 4 of the paraffin sections of 6 μ m～10 μ m, use QIAamp DNA FFPE Tissue Kit or extract genomic dna with the commercialization DNA extraction test kit of performance, concrete operation step is pressed test kit specification sheets.With spectrophotometer, the DNA storing solution after extracting is carried out to concentration determination, should at least reach 10ng/ μ L, and OD
260nm/ OD
280nmratio should be in 1.6-2.0 scope.The working fluid that DNA is diluted to 10ng/ μ L is for subsequent use.
2. detect BRAF transgenation
Above-mentioned prepared sample DNA and company standard product are carried out to following reaction system preparation:
A) preparation mixed solution (Mix), the preparation of ratio shown according to the form below.
| ? | Every reaction volume (μ L) | N reaction volume (μ L) |
| Detection reaction liquid | 20 | 20×N |
| Taq enzyme | 1 | 1×N |
B) mixed solution packing, adds 20 μ L Mix by the every hole/pipe of stoichiometric number.
C) add template, in positive control reacting hole/pipe, negative control reacting hole/pipe, sample reacting hole/pipe, add successively respectively in order 5 μ L positive controls, negative control, sample DNA (working fluid that the sample DNA suggestion that paraffin section extracts is diluted to 10ng/ μ L uses).
D) in reaction system, each component final concentration is as follows:
Wherein, the component of reaction system comprises primer and the probe as shown in SEQ ID:1～SEQ ID:7, synthetic by Sangon Biotech (Shanghai) Co., Ltd..
E) real-time PCR reactions condition is as follows:
Reaction volume: 25 μ L; The denaturation stage: 95 DEG C 10 minutes, 1 circulation; The amplification stage: 95 DEG C 30 seconds, 61 DEG C 1 minute, 50 circulations; Signal collection: collect FAM and HEX (or VIC) signal in the time of 61 DEG C of amplification stages.
3. detected result judgement
Detect FAM and HEX passage fluorescent signal Ct value, according to Ct value judged result: detect FAM and HEX passage amplification curve, reaching setting threshold (Ct is less than 30) with HEX signal is to show that DNA effective content is in allowed band, FAM channel signal credible result; While reaching the threshold value of setting using FAM channel signal, needed Ct value is as yin and yang attribute criterion: Ct < 48, and pattern detection is positive; Ct >=48 or without amplification, the negative or mutant nucleotide sequence copy number of pattern detection is lower than the detection lower limit of test kit.
Specific test: BRAF gene V600 wild-type clinical sample is all without FAM passage amplified signal, only have containing the template FAM passage of mutant plasmid and just have amplified signal, proved specificity of the present invention (seeing Fig. 1 (A)-Fig. 1 (D)).
Replica test: detect with a high mutation rate or low mutation rate standard substance, repeat imprecision CV≤5% 20 times.
Sensitivity test: detect the standard substance that under 50ng people's wild type gene group DNA background, mutation rate is 1%, can reach 95% positive rate.
Embodiment 2
In the clinical trial of 3 front three hospitals, adopting current transgenation gold standard detection method---Sanger method gene sequencing in contrast, has been carried out clinical experimental study to test kit of the present invention (being the test kit of embodiment 1).
Clinical trial detects altogether clinical samples and exceedes thousand examples, and tumor type comprises thyroid carcinoma, melanoma and colorectal cancer.The sensitivity (positive coincidence rate) of test kit of the present invention is 97.4%, and specific degree (negative match-rate) is 90.6%.Accuracy (total concordance rate) is 93.5%.Through Kappa inspection, two kinds of method consistence are excellent.To sum up analyze, whether mankind BRAF detection in Gene Mutation test kit of the present invention (fluorescent PCR method) exists in BRAF transgenation and detects and have very high consistence with gold standard Sanger gene sequencing method at the clinical paraffin embedding tumor tissues of detection, and test kit of the present invention can meet clinical detection needs.
Embodiment 3
Get clinical paraffin embedding thyroid carcinoma sample 1 example of collecting in January, 2013, application the present invention carries out BRAF detection in Gene Mutation to it.
1.DNA extracts
Approximately 4 of paraffin sections getting thickness and be 6 μ m～10 μ m, use QIAamp DNA FFPE Tissue Kit or extract genomic dna with the commercialization DNA extraction test kit of performance, and concrete operation step is pressed test kit specification sheets.With spectrophotometer, the DNA storing solution after extracting is carried out to concentration determination, should at least reach 10ng/ μ L, and OD
260nm/ OD
280nmratio should be in 1.6-2.0 scope.The working fluid that DNA is diluted to 10ng/ μ L is for subsequent use.
2. detect BRAF transgenation
Above-mentioned prepared sample DNA is carried out to following reaction system preparation:
A) preparation mixed solution (Mix), the preparation of ratio shown according to the form below.
| ? | Every reaction volume (μ L) | N reaction volume (μ L) |
| Detection reaction liquid | 20 | 20×N |
| Taq enzyme | 1 | 1×N |
B) mixed solution packing, adds 20 μ L Mix by the every hole/pipe of stoichiometric number.
C) add template, in positive control reacting hole/pipe, negative control reacting hole/pipe, sample reacting hole/pipe, add successively respectively in order 5 μ L positive controls, negative control, sample DNA (working fluid that the sample DNA suggestion that paraffin section extracts is diluted to 10ng/ μ L uses).
D) in reaction system, each component final concentration is as follows:
Wherein, the component of reaction system comprises primer and the probe as shown in SEQ ID:1～SEQ ID:7, synthetic by Sangon Biotech (Shanghai) Co., Ltd..
E) real-time PCR reactions condition is as follows:
Reaction volume: 25 μ L; The denaturation stage: 95 DEG C 10 minutes, 1 circulation; The amplification stage: 95 DEG C 30 seconds, 61 DEG C 1 minute, 50 circulations; Signal collection: collect FAM and HEX (or VIC) signal in the time of 61 DEG C of amplification stages.
3. detected result judgement
Detect FAM and HEX passage fluorescent signal Ct value, according to Ct value judged result: detect FAM and HEX passage amplification curve, reaching setting threshold (Ct is less than 30) with HEX signal is to show that DNA effective content is in allowed band, FAM channel signal credible result; While reaching the threshold value of setting using FAM channel signal, needed Ct value is as yin and yang attribute criterion: Ct < 48, and pattern detection is positive; Ct >=48 or without amplification, the negative or mutant nucleotide sequence copy number of pattern detection is lower than the detection lower limit of test kit.
Detected result shows, this thyroid cancer patients BRAF gene V600E sudden change positive (seeing Fig. 2 (A) and Fig. 2 (B)).Based on above result, predict that this patient's cancerous tissue invasiveness is high, easily invade profit Tiroidina surrounding tissue, chemotherapy effect is poor, should carry out operative treatment, but postoperative cancerous tissue is residual and risk of recurrence is high.
Embodiment 4
Get clinical paraffin embedding melanoma sample 1 example of collecting in January, 2013, application the present invention carries out BRAF detection in Gene Mutation to it.
1.DNA extracts
Approximately 4 of paraffin sections getting thickness and be 6 μ m～10 μ m, use QIAamp DNA FFPE Tissue Kit or extract genomic dna with the commercialization DNA extraction test kit of performance, and concrete operation step is pressed test kit specification sheets.With spectrophotometer, the DNA storing solution after extracting is carried out to concentration determination, should at least reach 10ng/ μ L, and OD
260nm/ OD
280nmratio should be in 1.6-2.0 scope.The working fluid that DNA is diluted to 10ng/ μ L is for subsequent use.
2. detect BRAF transgenation
Above-mentioned prepared sample DNA is carried out to following reaction system preparation:
A) preparation mixed solution (Mix), the preparation of ratio shown according to the form below.
| ? | Every reaction volume (μ L) | N reaction volume (μ L) |
| Detection reaction liquid | 20 | 20×N |
| Taq enzyme | 1 | 1×N |
B) mixed solution packing, adds 20 μ L Mix by the every hole/pipe of stoichiometric number.
C) add template, in positive control reacting hole/pipe, negative control reacting hole/pipe, sample reacting hole/pipe, add successively respectively in order 5 μ L positive controls, negative control, sample DNA (working fluid that the sample DNA suggestion that paraffin section extracts is diluted to 10ng/ μ L uses).
D) in reaction system, each component final concentration is as follows:
Wherein, the component of reaction system comprises the primer pair described in any one and probe in claim 1-3, synthetic by Sangon Biotech (Shanghai) Co., Ltd..
E) real-time PCR reactions condition is as follows:
Reaction volume: 25 μ L; The denaturation stage: 95 DEG C 10 minutes, 1 circulation; The amplification stage: 95 DEG C 30 seconds, 61 DEG C 1 minute, 50 circulations; Signal collection: collect FAM and HEX (or VIC) signal in the time of 61 DEG C of amplification stages.
3. detected result judgement
Detect FAM and HEX passage fluorescent signal Ct value, according to Ct value judged result: detect FAM and HEX passage amplification curve, reaching setting threshold (Ct is less than 30) with HEX signal is to show that DNA effective content is in allowed band, FAM channel signal credible result; While reaching the threshold value of setting using FAM channel signal, needed Ct value is as yin and yang attribute criterion: Ct < 48, and pattern detection is positive; Ct >=48 or without amplification, the negative or mutant nucleotide sequence copy number of pattern detection is lower than the detection lower limit of test kit.
Detected result shows, this melanoma patients BRAF gene V600E sudden change positive (seeing Fig. 3 (A) and Fig. 3 (B)).Based on above result, this patient should select this type of inhibition from mutation agent for BRAF gene V600E of Vemurafenib.
Embodiment 5
Get clinical paraffin embedding colorectal cancer sample 1 example of collecting in January, 2013, application the present invention carries out BRAF detection in Gene Mutation to it.
1.DNA extracts
Approximately 4 of paraffin sections getting thickness and be 6 μ m～10 μ m, use QIAamp DNA FFPE Tissue Kit or extract genomic dna with the commercialization DNA extraction test kit of performance, and concrete operation step is pressed test kit specification sheets.With spectrophotometer, the DNA storing solution after extracting is carried out to concentration determination, should at least reach 10ng/ μ L, and OD
260nm/ OD
280nmratio should be in 1.6-2.0 scope.The working fluid that DNA is diluted to 10ng/ μ L is for subsequent use.
2. detect BRAF transgenation
Above-mentioned prepared sample DNA is carried out to following reaction system preparation:
A) preparation mixed solution (Mix), the preparation of ratio shown according to the form below.
| ? | Every reaction volume (μ L) | N reaction volume (μ L) |
| Detection reaction liquid | 20 | 20×N |
| Taq enzyme | 1 | 1×N |
B) mixed solution packing, adds 20 μ L Mix by the every hole/pipe of stoichiometric number.
C) add template, in positive control reacting hole/pipe, negative control reacting hole/pipe, sample reacting hole/pipe, add successively respectively in order 5 μ L positive controls, negative control, sample DNA (working fluid that the sample DNA suggestion that paraffin section extracts is diluted to 10ng/ μ L uses).
D) in reaction system, each component final concentration is as follows:
Wherein, the component of reaction system comprises primer and the probe as shown in SEQ ID:1～SEQ ID:7, synthetic by Sangon Biotech (Shanghai) Co., Ltd..
E) real-time PCR reactions condition is as follows:
Reaction volume: 25 μ L; The denaturation stage: 95 DEG C 10 minutes, 1 circulation; The amplification stage: 95 DEG C 30 seconds, 61 DEG C 1 minute, 50 circulations; Signal collection: collect FAM and HEX (or VIC) signal in the time of 61 DEG C of amplification stages.
3. detected result judgement
Detect FAM and HEX passage fluorescent signal Ct value, according to Ct value judged result: detect FAM and HEX passage amplification curve, reaching setting threshold (Ct is less than 30) with HEX signal is to show that DNA effective content is in allowed band, FAM channel signal credible result; While reaching the threshold value of setting using FAM channel signal, needed Ct value is as yin and yang attribute criterion: Ct < 48, and pattern detection is positive; Ct >=48 or without amplification, the negative or mutant nucleotide sequence copy number of pattern detection is lower than the detection lower limit of test kit.
Detected result shows, this colorectal cancer patients BRAF gene V600E sudden change positive (seeing Fig. 4 (A) and Fig. 4 (B)).Based on above result, point out this patient may have former resistance to EGFR targeted drug, use must appropriate (Cetuximab) or the curative effect of the targeted drug such as Victibix (Panitumumab) poor.
Should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although the present invention is explained in detail with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify or be equal to replacement technical scheme of the present invention, and not depart from essence and the scope of technical solution of the present invention.
Claims (7)
1. a mankind BRAF detection in Gene Mutation primer pair and probe, detect for fluorescent PCR, it is characterized in that, described primer comprise for the Auele Specific Primer of containing BRAF gene mutation site to the internal control primer pair of monitoring sample DNA quality, described Auele Specific Primer is to being:
B-AR-F：GTGATTTTGGTCTAGCTACACA，
B-AR-R：CTAGTAACTCAGCAGCATCTCA；
Described internal control primer pair is:
B-IC-F：ATTGTTACCCAGTGGTGTGA，
B-IC-R：TCGTGCAATATCTATAAGTTTGA；
Described probe comprises the internal control probe for the specific probe of BRAF gene mutation site and monitoring sample DNA quality, specific probe sequence is sequence as shown in SEQ ID:3 or the complementary sequence of this sequence, and internal control probe sequence is sequence as shown in SEQ ID:6 and SEQ ID:7 or the sequence with the sequence complementation shown in SEQ ID:6 and SEQ ID:7.
2. mankind BRAF detection in Gene Mutation primer pair according to claim 1 and probe, is characterized in that: described BRAF gene mutation site is the T → A base mutation occurring on 1799 Nucleotide of BRAF gene extron 15.
3. mankind BRAF detection in Gene Mutation primer pair according to claim 1 and probe, is characterized in that, described probe is fluorescence labeling probe, and the specific probe after fluorescent mark is:
B-AR-P：6-FAM-ACCCTGGAGTGGGTCCCATCAGTTTGGG-BHQ1；
Internal control probe after fluorescent mark is:
B-IC-P1：HEX-CAGCTTGTATCACCATCTCCATAT-NH2
B-IC-P2：GATATGGAGATGGTGATACAAGCT-Dabcyl。
4. a mankind BRAF detection in Gene Mutation test kit, detects for fluorescent PCR, it is characterized in that: described test kit comprises the primer pair described in any one and probe in claim 1-3.
5. mankind BRAF detection in Gene Mutation test kit according to claim 4, is characterized in that: described test kit also comprises positive control and negative control.
6. the test kit of mankind BRAF detection in Gene Mutation according to claim 4, is characterized in that, described test kit comprises reaction solution, is dissolved with described primer pair and probe in reaction solution, and reaction solution also comprises PCR damping fluid, dNTP mixed solution, MgCl
2, Taq enzyme and remove DNA enzyme distilled water.
7. the test kit of mankind BRAF detection in Gene Mutation according to claim 6, it is characterized in that, in described reaction solution, the reaction density of each component is: described Taq enzyme 0.1-0.5U/ μ L, each primer 0.1-0.5 μ M, each probe 0.1-0.5 μ M in primer pair, PCR damping fluid 1 ×, dNTP mixed solution 0.5-1mM, MgCl
22.0-5.0mM, go DNA enzyme distilled water to add to configuration 1 × reaction system cumulative volume.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410274543.2A CN104017887A (en) | 2014-06-18 | 2014-06-18 | Primer pair as well as probe and kit for detecting human BRAF gene mutation |
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| CN201410274543.2A CN104017887A (en) | 2014-06-18 | 2014-06-18 | Primer pair as well as probe and kit for detecting human BRAF gene mutation |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593490A (en) * | 2014-12-23 | 2015-05-06 | 复旦大学附属华山医院 | Application of USP8 gene detection object in preparation for ACTH-type pituitary adenoma molecular pathological diagnosis and typing products |
| CN105296666A (en) * | 2015-12-07 | 2016-02-03 | 湖南圣维基因科技有限公司 | Fluorescent PCR detecting kit for V600E mutation of BRAF gene |
| CN107050027A (en) * | 2017-04-28 | 2017-08-18 | 中国人民解放军第二军医大学第二附属医院 | A Pa replaces application of the Buddhist nun in BRAFV600E kinases inhibitors are prepared |
| CN108070653A (en) * | 2016-11-15 | 2018-05-25 | 江苏众红生物工程创药研究院有限公司 | Kit and its application for the detection of people's BRAF gene mutation |
| CN109234369A (en) * | 2018-10-21 | 2019-01-18 | 上海睿璟生物科技有限公司 | A kind of PCR kit detecting people's BRAF gene mutation |
| CN110951849A (en) * | 2019-12-30 | 2020-04-03 | 武汉光谷联合医学检验所股份有限公司 | PCR kit for detecting human B-raf gene mutation and application thereof |
| CN113403397A (en) * | 2021-07-15 | 2021-09-17 | 西安医臻生物医药科技有限公司 | Primer group and kit for detecting melanoma targeted medication related gene mutation |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102242207A (en) * | 2011-06-29 | 2011-11-16 | 浙江大学 | Primers and probes for detecting mutation of cancer gene BRAFV600E |
| CN102575295A (en) * | 2009-08-11 | 2012-07-11 | 应答遗传公司 | Methods, primers, probes and kits useful for the detection of braf mutations |
| CN102586401A (en) * | 2011-01-05 | 2012-07-18 | 苏州科贝生物技术有限公司 | Method and kit for detecting mutation of BRAF gene of human colorectal cancer |
| CN102816851A (en) * | 2012-08-29 | 2012-12-12 | 苏州旷远生物分子技术有限公司 | Primer, probe, reagent kit and method for detecting mutation of BRAF gene V600E |
| CN103571948A (en) * | 2013-10-09 | 2014-02-12 | 武汉康录生物技术有限公司 | Kit for detecting hotspot mutation of BRAF gene and detection method thereof |
-
2014
- 2014-06-18 CN CN201410274543.2A patent/CN104017887A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102575295A (en) * | 2009-08-11 | 2012-07-11 | 应答遗传公司 | Methods, primers, probes and kits useful for the detection of braf mutations |
| CN102586401A (en) * | 2011-01-05 | 2012-07-18 | 苏州科贝生物技术有限公司 | Method and kit for detecting mutation of BRAF gene of human colorectal cancer |
| CN102242207A (en) * | 2011-06-29 | 2011-11-16 | 浙江大学 | Primers and probes for detecting mutation of cancer gene BRAFV600E |
| CN102816851A (en) * | 2012-08-29 | 2012-12-12 | 苏州旷远生物分子技术有限公司 | Primer, probe, reagent kit and method for detecting mutation of BRAF gene V600E |
| CN103571948A (en) * | 2013-10-09 | 2014-02-12 | 武汉康录生物技术有限公司 | Kit for detecting hotspot mutation of BRAF gene and detection method thereof |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593490A (en) * | 2014-12-23 | 2015-05-06 | 复旦大学附属华山医院 | Application of USP8 gene detection object in preparation for ACTH-type pituitary adenoma molecular pathological diagnosis and typing products |
| CN105296666A (en) * | 2015-12-07 | 2016-02-03 | 湖南圣维基因科技有限公司 | Fluorescent PCR detecting kit for V600E mutation of BRAF gene |
| CN108070653A (en) * | 2016-11-15 | 2018-05-25 | 江苏众红生物工程创药研究院有限公司 | Kit and its application for the detection of people's BRAF gene mutation |
| CN107050027A (en) * | 2017-04-28 | 2017-08-18 | 中国人民解放军第二军医大学第二附属医院 | A Pa replaces application of the Buddhist nun in BRAFV600E kinases inhibitors are prepared |
| CN109234369A (en) * | 2018-10-21 | 2019-01-18 | 上海睿璟生物科技有限公司 | A kind of PCR kit detecting people's BRAF gene mutation |
| CN110951849A (en) * | 2019-12-30 | 2020-04-03 | 武汉光谷联合医学检验所股份有限公司 | PCR kit for detecting human B-raf gene mutation and application thereof |
| CN110951849B (en) * | 2019-12-30 | 2023-09-19 | 武汉光谷联合医学检验实验室股份有限公司 | PCR (polymerase chain reaction) kit for detecting human B-raf gene mutation and application thereof |
| CN113403397A (en) * | 2021-07-15 | 2021-09-17 | 西安医臻生物医药科技有限公司 | Primer group and kit for detecting melanoma targeted medication related gene mutation |
| CN113403397B (en) * | 2021-07-15 | 2024-03-08 | 西安医臻生物医药科技有限公司 | Primer set and kit for detecting melanoma targeted drug-related gene mutation |
| CN114634987A (en) * | 2022-04-21 | 2022-06-17 | 北京积水潭医院 | Primer probe composition and kit for detecting BRAF gene mutation, and use method and application thereof |
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Application publication date: 20140903 |