CN112029832A - Method for rapidly detecting BRAF-KIAA1549 fusion mutation - Google Patents
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Abstract
The invention discloses a method for rapidly detecting BRAF-KIAA1549 fusion mutation. The invention uses the fused RNA of BRAF-KIAA1549 as the initial template of the reaction, effectively avoids the interference of genome DNA and ensures that the detection result is more accurate; the reverse transcription and the real-time PCR share a reaction system, compared with the traditional reverse transcription, the detection is carried out after the amplification by taking cDNA as a template, the steps are more simplified, and the pollution possibly caused by opening the cover for many times is avoided; the housekeeping gene ACTB is introduced into a single system through the change of the fluorescent group, so that the existence of negative control is ensured, and the risk of false negative is reduced; the result is interpreted by the delta Ct, so that the risk of false positive is reduced, and the experimental result is more accurate and credible; the designed PCR product is relatively small, so that the paraffin-embedded sample with high degradation degree can also meet the detection requirement. The detection method disclosed by the invention is simple to operate, effectively avoids redundant steps and possible pollution, and simultaneously ensures the reliability and accuracy of the detection result by adding contrast.
Description
Technical Field
The invention relates to the field of cancer detection and molecular biology, in particular to a method for quickly detecting BRAF-KIAA1549 fusion mutation.
Background
Malignant tumor is one of the diseases which seriously affect human health at present, and the internal cause of the malignant tumor is that the cell growth regulation is abnormal due to the change of genes in cells, so that abnormal hyperplasia is caused, and then the tumor is generated. The BRAF gene is an important transfer factor of a Ras-Raf-MEK-ERK signal pathway and is one of the most common mutant genes in human cancers. The Ras-Raf-MEK-ERK signal pathway is a Mitogen-Activated Protein Kinase (MAPK) pathway which can be widely Activated, and participates in various biological reactions such as cell proliferation and differentiation, cell morphology maintenance, cytoskeleton construction, cell apoptosis, cell malignant change and the like through serine/threonine Protein Kinase in a serine Protein Kinase pathway.
The BRAF gene can be rearranged with various genes and plays an important role in the development of tumors, wherein KIAA1549-BRAF fusion mutation can occur in 60-80% of hair cell astrocytomas. In the fusion mutation of BRAF-KIAA1549, the self-inhibition domain at the N terminal of BRAF is replaced by KIAA1549, while the C terminal domain of BRAF is reserved, so that the kinase activity is retained, and the MAPK pathway is abnormally activated, thereby causing tumorigenesis. And the conventional BRAF fragmentation probe and the BRAF-KIAA1549 fusion gene probe cannot completely distinguish positive samples from negative samples. Therefore, the invention provides a method for rapidly detecting the fusion mutation of BRAF-KIAA 1549.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a method for quickly detecting the fusion mutation of BRAF-KIAA 1549.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for rapidly detecting BRAF-KIAA1549 fusion mutation comprises the following steps:
(1) extracting RNA of the tumor tissue;
(2) constructing a PCR reaction system;
(3) performing real-time fluorescent quantitative PCR by a TaqMan probe method;
(4) and (5) judging the result.
The method for rapidly detecting the fusion mutation of BRAF-KIAA1549 comprises the following steps of (2): and (2) taking the RNA extracted in the step (1) as a template, adding reverse transcriptase, polymerase, BRAF-KIAA1549 upstream and downstream primers, a BRAF-KIAA1549TaqMan probe, an ACTB upstream and downstream primer and an ACTB TaqMan probe into the same reaction system, and changing the reaction temperature of the PCR reaction system to realize three processes of cDNA construction, PCR product amplification and fluorescent signal collection.
The method for rapidly detecting the fusion mutation of BRAF-KIAA1549 comprises the following steps of (4): determining whether the sample is positive for BRAF-KIAA1549 mutation or not according to the numerical value of the delta Ct, wherein the delta Ct is Ct (BRAF-KIAA1549) -Ct (ACTB); if delta Ct is more than 0 and less than 10, the sample is positive in BRAF-KIAA1549 mutation; increasing the dosage of RNA and detecting again, wherein the dosage of the delta Ct is 10; delta Ct is more than 10 or no BRAF-KIAA1549 specific fluorescent new number is collected, and the sample is BRAF-KIAA1549 negative.
The sequence of the upstream primer of the BRAF-KIAA1549 is 5 'AGTGGGGGTCCTTCTACAGC-3', the sequence of the downstream primer of the BRAF-KIAA1549 is 5 'TCCTCCATCACCACGAAATCC-3', and the sequence of the BRAF-KIAA1549TaqMan probe is 5 '-FAM-CAGCCCAGACGGCCAA-BHQ 1-3'; the sequence of the ACTB upstream primer is 5 'CTTCGCGGGCGACGAT-3', the sequence of the ACTB downstream primer is 5 'ATAGGAATCCTTCTGACCCATGC-3', and the sequence of the ACTB TaqMan probe is 5 '-HEX-CGGGCCGTCTTCCC-BHQ 1-3'.
The invention has the beneficial effects that: (1) the invention uses the fused RNA of BRAF-KIAA1549 as the initial template of the reaction, effectively avoids the interference of genome DNA and ensures that the detection result is more accurate; (2) the reverse transcription and the real-time PCR share a reaction system to complete the cDNA construction with RNA as a template, the PCR amplification with cDNA as a template and the detection of products by a Taqman probe. Compared with the traditional method of detecting after reverse transcription and cDNA as a template amplification, the method has more simplified steps. A single system is constructed, so that the pollution possibly caused by multiple uncovering is avoided; (3) the invention adopts Taqman real-time PCR to detect the BRAF-KIAA1549 fusion mutation condition, and compared with agarose gel electrophoresis, the invention avoids the pollution possibly caused by uncovering; compared with SYBR Green real-time PCR method, the Taqman specific probe is more sensitive and accurate; (4) in the invention, the housekeeping gene ACTB is introduced into a single system through the change of the fluorescent group, so that the existence of negative control is ensured, and the risk of false negative is reduced; the result is interpreted by the delta Ct, so that the risk of false positive is reduced, and the experimental result is more accurate and credible; (5) the PCR product designed by the invention is relatively small, so that the paraffin embedded sample with high degradation degree can also meet the detection requirement. In conclusion, the detection method is simple to operate, redundant steps and possible pollution are effectively avoided, and the reliability and accuracy of the detection result are ensured by adding contrast while the detection is simple.
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The invention is further illustrated with reference to the following figures and examples.
FIG. 1 is a TaqMan real-time PCR amplification profile of sample 1 in example 1;
FIG. 2 is a sample 2TaqMan real-time PCR amplification profile in example 2.
Detailed Description
In order to make the technical solutions of the present invention better understood, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
[ example 1 ]
A method for rapidly detecting BRAF-KIAA1549 fusion mutation comprises the following steps:
(1) extracting RNA of tumor tissue of the sample 1;
(2) constructing a PCR reaction system;
(3) performing real-time fluorescent quantitative PCR by a TaqMan probe method;
(4) and (5) judging the result.
Further, the extraction procedure for sample 1RNA was as follows: dewaxing a paraffin sample: 5 paraffin tissue sections of 7 μm samples 1 were placed in a 1.5ml centrifuge tube, 1ml xylene was added, centrifugation was carried out at 13,000Xg for 1 minute, the supernatant was discarded, 1ml xylene was added, centrifugation was carried out at 13,000Xg for 1 minute, and the supernatant was discarded. Adding 1ml of absolute ethyl alcohol into a centrifuge tube, centrifuging for 1 minute at 13,000Xg, removing supernatant, and airing; adding tissue lysate and proteinase K into the air-dried tissue, splitting at 56 ℃ for 15 minutes, transferring the centrifuge tube to 80 ℃, keeping for 15 minutes, taking down, adding DNase into the centrifuge tube after the centrifuge tube is moved to room temperature, and standing for 15 minutes at room temperature; adding a binding buffer solution and absolute ethyl alcohol into a sample, gently mixing uniformly, transferring the sample to an RNA extraction filter column, centrifuging for 1 minute at 13,000Xg, adding 600 mu L of a washing buffer solution into the sample, centrifuging for 1 minute at 13,000Xg, and repeatedly washing for three times; putting the filter column into a new 1.5ml centrifuge tube, placing the centrifuge tube at 56 ℃, opening the cover, airing for 3 minutes, adding eluent, covering the cover, and keeping at 56 ℃ for 2 minutes; the sample 1RNA sample was obtained by centrifugation at 13,000Xg for 1 minute.
Further, the PCR reaction system is constructed as follows: firstly, 2 mu L of RNA is taken as a template for constructing cDNA, and a housekeeping gene ACTB is introduced into a reaction system, so that the existence of negative control is ensured, the risk of false positive is reduced, the result interpretation is facilitated, and the specific reaction system comprises the following components:
the three processes of cDNA construction, PCR product amplification and fluorescent signal collection are realized by changing the reaction temperature of a reaction system, and the specific reaction program is as follows:
and after the reaction system is constructed, performing TaqMan real-time PCR to detect the fusion mutation condition of BRAF-KIAA 1549.
Further, the step (4) is specifically as follows: determining whether the sample is positive for BRAF-KIAA1549 mutation or not according to the numerical value of the delta Ct, wherein the delta Ct is Ct (BRAF-KIAA1549) -Ct (ACTB); if delta Ct is more than 0 and less than 10, the sample is positive in BRAF-KIAA1549 mutation; increasing the dosage of RNA and detecting again, wherein the dosage of the delta Ct is 10; delta Ct is more than 10 or no BRAF-KIAA1549 specific fluorescent new number is collected, and the sample is BRAF-KIAA1549 negative.
Further, the sequence of the BRAF-KIAA1549 upstream primer is 5 'AGTGGGGGTCCTTCTACAGC-3', the sequence of the BRAF-KIAA1549 downstream primer is 5 'TCCTCCATCACCACGAAATCC-3', and the sequence of the BRAF-KIAA1549TaqMan probe is 5 '-FAM-CAGCCCAGACGGCCAA-BHQ 1-3'; the sequence of the ACTB upstream primer is 5 'CTTCGCGGGCGACGAT-3', the sequence of the ACTB downstream primer is 5 'ATAGGAATCCTTCTGACCCATGC-3', and the sequence of the ACTB TaqMan probe is 5 '-HEX-CGGGCCGTCTTCCC-BHQ 1-3'.
The amplification map obtained by the sample 1TaqMan real-time PCR of the present example is shown in FIG. 1: ● in FIG. 1 represents the HEX fluorescence signal, the negative control ACTB; ■ represents the FAM fluorescence signal, BRAF-KIAA1549 fusion mutation, depicted in fig. 1, Ct (actb) 17.0, Ct (BRAF-KIAA1549 fusion) 18.1, Δ Ct 1.1, thus the fig. 1 sample was positive for the BRAF-KIAA1549 fusion mutation.
[ example 2 ]
A method for rapidly detecting BRAF-KIAA1549 fusion mutation comprises the following steps:
(1) extracting RNA of tumor tissue of the sample 2;
(2) constructing a PCR reaction system;
(3) performing real-time fluorescent quantitative PCR by a TaqMan probe method;
(4) and (5) judging the result.
Further, the extraction procedure for RNA from sample 2 was as follows: dewaxing a paraffin sample: 5 samples (7 μm) of 2 paraffin tissue sections were taken and placed in a 1.5ml centrifuge tube, 1ml of xylene was added, centrifugation was carried out at 13,000Xg for 1 minute, the supernatant was discarded, 1ml of xylene was added, centrifugation was carried out at 13,000Xg for 1 minute, and the supernatant was discarded. Adding 1ml of absolute ethyl alcohol into a centrifuge tube, centrifuging for 1 minute at 13,000Xg, removing supernatant, and airing; adding tissue lysate and proteinase K into the air-dried tissue, splitting at 56 ℃ for 15 minutes, transferring the centrifuge tube to 80 ℃, keeping for 15 minutes, taking down, adding DNase into the centrifuge tube after the centrifuge tube is moved to room temperature, and standing for 15 minutes at room temperature; adding a binding buffer solution and absolute ethyl alcohol into a sample, gently mixing uniformly, transferring the sample to an RNA extraction filter column, centrifuging for 1 minute at 13,000Xg, adding 600 mu L of a washing buffer solution into the sample, centrifuging for 1 minute at 13,000Xg, and repeatedly washing for three times; putting the filter column into a new 1.5ml centrifuge tube, placing the centrifuge tube at 56 ℃, opening the cover, airing for 3 minutes, adding eluent, covering the cover, and keeping at 56 ℃ for 2 minutes; the sample 2RNA sample was obtained by centrifugation at 13,000Xg for 1 minute.
Further, the PCR reaction system is constructed as follows: firstly, 2 mu L of RNA is taken as a template for constructing cDNA, and a housekeeping gene ACTB is introduced into a reaction system, so that the existence of negative control is ensured, the risk of false positive is reduced, the result interpretation is facilitated, and the specific reaction system comprises the following components:
| 20 μ L reverse transcription system component | Volume (μ L) |
| 2×One Step RT-PCR BufferⅢ(Takara) | 10 |
| TaKaRa Ex Taq HS(5U/μL)(Takara) | 0.4 |
| PrimeScript RT Enzyme MixⅡ(Takara) | 0.4 |
| BRAF-KIAA1549 upstream primer (10. mu.M) | 0.4 |
| BRAF-KIAA1549 downstream primer (10. mu.M) | 0.4 |
| BRAF-KIAA1549TaqMan probe | 0.8 |
| ACTB upstream primer (10. mu.M) | 0.4 |
| ACTB downstream primer (10. mu.M) | 0.4 |
| ACTB TaqMan probes | 0.8 |
| |
2 |
| RNase Free dH2O | 4 |
| |
20 |
The three processes of cDNA construction, PCR product amplification and fluorescent signal collection are realized by changing the reaction temperature of a reaction system, and the specific reaction program is as follows:
and after the reaction system is constructed, performing TaqMan real-time PCR to detect the fusion mutation condition of BRAF-KIAA 1549.
Further, the step (4) is specifically as follows: determining whether the sample is positive for BRAF-KIAA1549 mutation or not according to the numerical value of the delta Ct, wherein the delta Ct is Ct (BRAF-KIAA1549) -Ct (ACTB); if delta Ct is more than 0 and less than 10, the sample is positive in BRAF-KIAA1549 mutation; increasing the dosage of RNA and detecting again, wherein the dosage of the delta Ct is 10; delta Ct is more than 10 or no BRAF-KIAA1549 specific fluorescent new number is collected, and the sample is BRAF-KIAA1549 negative.
Further, the sequence of the BRAF-KIAA1549 upstream primer is 5 'AGTGGGGGTCCTTCTACAGC-3', the sequence of the BRAF-KIAA1549 downstream primer is 5 'TCCTCCATCACCACGAAATCC-3', and the sequence of the BRAF-KIAA1549TaqMan probe is 5 '-FAM-CAGCCCAGACGGCCAA-BHQ 1-3'; the sequence of the ACTB upstream primer is 5 'CTTCGCGGGCGACGAT-3', the sequence of the ACTB downstream primer is 5 'ATAGGAATCCTTCTGACCCATGC-3', and the sequence of the ACTB TaqMan probe is 5 '-HEX-CGGGCCGTCTTCCC-BHQ 1-3'.
The amplification map obtained by TaqMan real-time PCR of sample 2 in this example is shown in FIG. 2: ● in FIG. 2 represents the HEX fluorescence signal, the negative control ACTB, and no FAM signal was collected, i.e., no amplification of the BRAF-KIAA1549 fusion mutation, so this sample was a wild sample.
The above embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and the scope of the present invention is defined by the claims. Various modifications and equivalents may be made by those skilled in the art within the spirit and scope of the present invention, and such modifications and equivalents should also be considered as falling within the scope of the present invention.
Claims (4)
1. A method for rapidly detecting BRAF-KIAA1549 fusion mutation is characterized by comprising the following steps:
(1) extracting RNA of the tumor tissue;
(2) constructing a PCR reaction system;
(3) performing real-time fluorescent quantitative PCR by a TaqMan probe method;
(4) and (5) judging the result.
2. The method for rapidly detecting the BRAF-KIAA1549 fusion mutation according to claim 1, wherein the step (2) is specifically as follows: taking the RNA extracted in the step (1) as a template, and adding reverse transcriptase, polymerase, BRAF-KIAA1549 upstream and downstream primers, a BRAF-KIAA1549TaqMan probe, an ACTB upstream and downstream primer and an ACTB TaqMan probe into the same reaction system; the three processes of cDNA construction, PCR product amplification and fluorescent signal collection are realized by changing the reaction temperature of a PCR reaction system.
3. The method for rapidly detecting the BRAF-KIAA1549 fusion mutation according to claim 1, wherein the step (4) is specifically as follows: determining whether the sample is positive for BRAF-KIAA1549 mutation or not according to the numerical value of the delta Ct, wherein the delta Ct is Ct (BRAF-KIAA1549) -Ct (ACTB); if delta Ct is more than 0 and less than 10, the sample is positive in BRAF-KIAA1549 mutation; increasing the dosage of RNA and detecting again, wherein the dosage of the delta Ct is 10; delta Ct is more than 10 or no BRAF-KIAA1549 specific fluorescent new number is collected, and the sample is BRAF-KIAA1549 negative.
4. The method for rapidly detecting the fusion mutation of BRAF-KIAA1549 as claimed in claim 2, wherein the sequence of the upstream primer of BRAF-KIAA1549 is 5 'AGTGGGGGTCCTTCTACAGC-3', the sequence of the downstream primer of BRAF-KIAA1549 is 5 'TCCTCCATCACCACGAAATCC-3', and the sequence of the BRAF-KIAA1549TaqMan probe is 5 '-FAM-CAGCCCAGACGGCCAA-BHQ 1-3'; the sequence of the ACTB upstream primer is 5 'CTTCGCGGGCGACGAT-3', the sequence of the ACTB downstream primer is 5 'TAGGAATCCTTCTGACCCATGC-3', and the sequence of the ACTB TaqMan probe is 5 '-HEX-CGGGCCGTCTTCCC-BHQ 1-3'.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109234400A (en) * | 2018-11-13 | 2019-01-18 | 武汉康录生物技术股份有限公司 | It is a kind of for detecting the fluorescence in situ hybridization probe and its preparation method and application of KIAA1549-BRAF fusion |
| CN110195098A (en) * | 2019-05-10 | 2019-09-03 | 广州安必平医药科技股份有限公司 | For detecting probe groups, kit and its application of BRAF correlation fusion gene |
| CN110863053A (en) * | 2019-12-18 | 2020-03-06 | 广州迈景基因医学科技有限公司 | Primer, probe and method for detecting EGFR vIII mutant |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109234400A (en) * | 2018-11-13 | 2019-01-18 | 武汉康录生物技术股份有限公司 | It is a kind of for detecting the fluorescence in situ hybridization probe and its preparation method and application of KIAA1549-BRAF fusion |
| CN110195098A (en) * | 2019-05-10 | 2019-09-03 | 广州安必平医药科技股份有限公司 | For detecting probe groups, kit and its application of BRAF correlation fusion gene |
| CN110863053A (en) * | 2019-12-18 | 2020-03-06 | 广州迈景基因医学科技有限公司 | Primer, probe and method for detecting EGFR vIII mutant |
Non-Patent Citations (1)
| Title |
|---|
| YONGJI TIAN等: "Detection of KIAA1549-BRAF fusion transcripts in formalin-fixed paraffin-embedded pediatric low-grade gliomas", 《J MOL DIAGN》, vol. 13, no. 6, pages 669 - 77 * |
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