CN113136419A - Fluorescent quantitative PCR detection method for fusion gene mutation - Google Patents
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Abstract
The invention discloses a fluorescent quantitative PCR detection method for fusion gene mutation, which comprises the following steps: 1) searching fusion gene sites; 2) fusion mutation sequences are subjected to merging analysis; 3) designing and synthesizing an amplification primer; 4) and amplifying and verifying the reaction system. The invention belongs to the technical field of biological detection, and provides a fluorescent quantitative PCR detection method of fusion gene mutation, which can design corresponding primers aiming at different fusion genes, realize the detection of the fusion gene mutation and improve the detection efficiency and the accuracy of a detection result.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a fluorescent quantitative PCR detection method for fusion gene mutation.
Background
A fusion gene is a chimeric gene formed by connecting the coding regions of two or more genes end to end and placing them under the control of the same set of regulatory sequences (including promoter, enhancer and ribosome binding sequence). The expression product of the fusion gene is fusion protein, and the mutation of the fusion gene can cause the abnormality of protein level, function and action site, and easily cause or promote the generation of tumor.
The detection method aiming at the mutation of the fusion gene mainly comprises the following steps: immunohistochemistry (IHC), Fluorescence In Situ Hybridization (FISH), and High-throughput sequencing. The immunohistochemical method is a protein detection method based on tissues, tumor cells subjected to gene fusion have obvious membrane staining reaction, the operation is simple and convenient, but the detection result is easily influenced, the result judgment has difference of subjective judgment, and false positive and false negative are easily generated. Fluorescence In Situ Hybridization (FISH) is used for joining a probe with fluorescent substances with target DNA by a special means according to the base complementary pairing principle, and finally, the position of the target DNA can be directly observed by using a Fluorescence microscope; the method has high detection sensitivity, but is complex to operate, plane reading needs to be carried out on a carrier, a three-dimensional space reading function is lacked, misjudgment possibly caused by space folding exists, manual interpretation is needed, the method is limited by the experience, complexity and the like of the manual interpretation, the interpretation has the problems of high technical requirements, deviation easily existing in interpretation results and the like, the requirement on storage of a detection sample is high, false negative easily occurs due to overlong retention time, and the method is limited to a certain extent in clinical detection. High-throughput sequencing technology (High-throughput sequencing) marks that sequence determination and general short reading length can be performed on hundreds of thousands to millions of DNA molecules at a time in parallel, has High requirements on related software, High sequencing cost, complex operation, long sequencing data reading time, insufficient accuracy and timeliness of low-frequency mutation detection results, and is difficult to be widely applied to clinical detection.
The two genes, EML4 and ALK, are located in the p21 and p23 bands of human chromosome 2, respectively, at a distance of about 10Mb apart. The inverted fusion of these two gene segments enables tissues to express the novel fusion protein. Different EML4-ALK fusion mutants often have malignant transformation and tumorigenicity capabilities, however, the detection method of EMI4-ALK fusion gene mutation still needs to improve the specificity and sensitivity of detection.
The fluorescent quantitative PCR detection technology is a new nucleic acid quantitative technology developed by PE company in America, is widely applied to aspects of gene expression difference analysis, SNP detection, prenatal diagnosis, drug efficacy assessment and the like, but is not widely applied to the aspect of fusion gene detection, so that the fluorescent quantitative PCR detection method for fusion gene mutation is provided, the detection efficiency and the detection result are improved, and the fluorescent quantitative PCR detection method has important significance.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a fluorescent quantitative PCR detection method for fusion gene mutation, which improves the detection efficiency and the accuracy of the detection result. The invention can design corresponding primers aiming at different fusion genes and realize the detection of the mutation of the fusion genes.
The objects of the invention will be further illustrated by the following detailed description.
The invention provides a fluorescent quantitative PCR detection method for fusion gene mutation, which comprises the following steps:
1) searching fusion gene sites: searching fusion gene information, searching exon fracture points, and searching transcript sequences from the exon fracture points to termination points;
2) fusion mutation sequences were analyzed in combination: merging the two sections of transcript sequences obtained in the step 1) to be used as a complete fusion result sequence;
3) designing and synthesizing an amplification primer: taking the fusion result sequence obtained in the step 2) as a sequence template, carrying out sequence design, synthesis and purification on upstream and downstream amplification primers;
4) amplification and verification of a reaction system: the method comprises the following steps: 4.1 preparing negative and positive RNA samples, diluting the positive RNA sample into a positive RNA sample with low mutation frequency according to a multiple proportion, and using the negative sample as a diluent in the dilution process; 4.2 reverse transcription of RNA sample, using the kit to carry out reverse transcription of RNA into cDNA; 4.3 reaction system preparation and PCR amplification: preparing a reaction system containing the upstream and downstream amplification primers, carrying out PCR amplification, and collecting a fluorescence signal after each cycle is finished; 4.4 judging the result: and reading the Tm value according to the amplification and dissolution curve, and judging the detection result.
Preferably, the fusion gene is EML4-ALK, and the exon fracture points thereof comprise an EML4 gene exon20 fracture point and an ALK gene exon20 fracture point.
Preferably, the upstream and downstream amplification primers comprise: upstream primer TGGTCCCCAGACAACAAGTAT, SEQ ID NO: 1, downstream primer TTGGGGTTGTAGTCGGTCAT, SEQ ID NO: 2.
preferably, the reaction system comprises the upstream and downstream amplification primers, an amplification reaction solution, cDNA and nuclease-free water; the PCR amplification operating conditions include: 30s at 95 ℃; 10s at 95 ℃, 30s at 60 ℃ and 40 cycles.
Preferably, the kit is a cDNA one-strand synthesis kit.
Compared with the prior art, the invention has the beneficial effects that:
(1) the fluorescent quantitative PCR detection method provided by the invention can design corresponding primers aiming at different fusion genes, realize the detection of the mutation of the fusion genes, improve the detection efficiency and have strong reliability of the detection result.
(2) The fluorescence quantitative PCR detection method provided by the invention has the advantages of rapid detection, simple and convenient operation, simple and visual result interpretation, and compared with FISH fluorescence in-situ hybridization, the interpretation level of technical personnel does not need to be too high, and the negative and positive judgments corresponding to the peak diagram change of the dissolution curve can be visually seen.
(3) The fluorescent quantitative PCR detection method provided by the invention does not need deeper manual analysis, and compared with a high-throughput sequencing technology, the fluorescent quantitative PCR detection method does not need bioinformatics process construction and analysis, so that the time is saved (generally, the high-throughput sequencing experiment process needs to comprise extraction, library construction, quality inspection quantification, sequencing on a computer and other processes, and needs to be completed in 3-5 days).
Drawings
FIG. 1 amplification and dissolution curves of negative samples.
FIG. 2 amplification lysis curve of positive sample.
FIG. 32 dilution the amplification lysis curve of positive samples.
FIG. 410 is an amplification and dissolution curve of a diluted positive sample.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Example 1 fluorescent quantitative PCR detection method of EML4-ALK fusion Gene mutation
The fluorescent quantitative PCR detection method of the EML4-ALK fusion gene mutation comprises the following steps:
1) searching fusion gene sites: searching for EML4-ALK fusion information in COSMIC (https:// cancer. sanger. ac. uk/COSMIC), searching for EML4 gene exon20 breakpoint, ALK gene exon20 breakpoint, and searching for the transcript sequence from EML4 gene origin to breakpoint and the transcript sequence from ALK gene breakpoint to termination point on NCBI (https:// www.ncbi.nlm.nih.gov /) according to exon breakpoint;
the sequences before and after the partial break point of EML4 exon20 are shown below (the deletion line is marked as the break point):
ATGGCTTCCAAATAGAAGTACAGGGTCATACAGATGAGCTTTGGGGTCTTGCCACACATCCCTTCAAAGATTTGCTCTTGACATGTGCTCAGGACAGGCAGGTGTGCCTGTGGAACTCAATGGAACACAGGCTGGAATGGACCAGGCTGGTAGATGAACCAGGACACTGTGCAGATTTTCATCCAAGTGGCACAGTGGTGGCCATAGGAACGCACTCAGGCAGGTGGTTTGTTCTGGATGCAGAAACCAGAGATCTAGTTTCTATCCACACAGACGGGAATGAACAGCTCTCTGTGATGCGCTACTCAATAGATGGTACCTTCCTGGCTGTAGGATCTCATGACAACTTTATTTACCTCTATGTAGTCTCTGAAAATGGAAGAAAATATAGCAGATATGGAAGGTGCACTGGACATTCCAGCTACATCACACACCTTGACTGGTCCCCAGACAACAAGTATATAATGTCTAACTCGGGAGACTATGAAATATTGTACTGGGACATTCCAAATGGCTGCAAACTAATCAGGAATCGATCGGATTGTAAGGACATTGATTGGACGACATATACCTGTGTGCTAGGATTTCAAGTATTTGGTGTCTGGCCAGAAGGATCTGATGGGACAGATATCAATGCACTGGTGCGATCCCACAATAGAAAGGTGATAGCTGTTGCCGATGACTTTTGTAAAGTCCATCTGTTTCAGTATCCCTGCTCCAAAGCAAAGGCTCCCAGTCACAAGTACAGTGCCCACAGCAGCCATGTCACCAATGTCAGTTTTACTCACAATGACAGTCACCTGATATCAACTGGTGGAAAAGACATGAGCATCATTCAGTGGAAACTTGTGGAAAAGTTATCTTTGCCTCAGAATGAGACTGTAGCGGATACTACTCTAACCAAAGCCCCCGTCTCTTCCACTGAAAGTGTCATCCAATCTAATACTCCCACACCGCCTCCTTCTCAGCCCTTAAATGAGACAGCTGAAGAGGAAAGTAG。
the sequence before and after the partial break point of ALK exon20 is shown below (deletion line labeled break site):
GATAACACTTCCTTGCTCTGGGCCGGAAAATCTTTGCAGGAGGGTGCCACCGGAGGACATTCCTGCCCCCAGGCCATGAAGAAGTGGGGGTGGGAGACAAGAGGGGGTTTCGGAGGGGGTGGAGGGGGGTGCTCCTCAGGTGGAGGAGGCGGAGGATATATAGGCGGCAATGCAGCCTCAAACAATGACCCCGAAATGGATGGGGAAGATGGGGTTTCCTTCATCAGTCCACTGGGCATCCTGTACACCCCAGCTTTAAAAGTGATGGAAGGCCACGGGGAAGTGAATATTAAGCATTATCTAAACTGCAGTCACTGTGAGGTAGACGAATGTCACATGGACCCTGAAAGCCACAAGGTCATCTGCTTCTGTGACCACGGGACGGTGCTGGCTGAGGATGGCGTCTCCTGCATTGTGTCACCCACCCCGGAGCCACACCTGCCACTCTCGCTGATCCTCTCTGTGGTGACCTCTGCCCTCGTGGCCGCCCTGGTCCTGGCTTTCTCCGGCATCATGATTGTGTACCGCCGGAAGCACCAGGAGCTGCAAGCCATGCAGATGGAGCTGCAGAGCCCTGAGTACAAGCTGAGCAAGCTCCGCACCTCGACCATCATGACCGACTACAACCCCAACTACTGCTTTGCTGGCAAGACCTCCTCCATCAGTGACCTGAAGGAGGTGCCGCGGAAAAACATCACCCTCATTCGGGGTCTGGGCCATGGCGCCTTTGGGGAGGTGTATGAAGGCCAGGTGTCCGGAATGCCCAACGACCCAAGCCCCCTGCAAGTGGCTGTGAAGACGCTGCCTGAAGTGTGCTCTGAACAGGACGAACTGGATTTCCTCATGGAAGCCCTGATCATCAGCAAATTCAACCACCAGAACATTGTTCGCTGCATTGGGGTGAGCCTGCAATCCCTGCCCCGGTTCATCCTGCTGGAGCTCATGGCGGGGGGAGACCTCAAGTCCTTCCTCCGAGAGACCCGCCCTCGCCCGAGCCAGC。
2) fusion mutation sequences were analyzed in combination: merging the two sections of transcript sequences obtained in the step 1), and taking the merged transcript sequences as a complete fusion result sequence as shown below (a deleted line is marked as a fracture site, and an underline is marked as an upstream and downstream complementary pairing site);
ATGGCTTCCAAATAGAAGTACAGGGTCATACAGATGAGCTTTGGGGTCTTGCCACACATCCCTTCAAAGATTTGCTCTTGACATGTGCTCAGGACAGGCAGGTGTGCCTGTGGAACTCAATGGAACACAGGCTGGAATGGACCAGGCTGGTAGATGAACCAGGACACTGTGCAGATTTTCATCCAAGTGGCACAGTGGTGGCCATAGGAACGCACTCAGGCAGGTGGTTTGTTCTGGATGCAGAAACCAGAGATCTAGTTTCTATCCACACAGACGGGAATGAACAGCTCTCTGTGATGCGCTACTCAATAGATGGTACCTTCCTGGCTGTAGGATCTCATGACAACTTTATTTACCTCTATGTAGTCTCTGAAAATGGAAGAAAATATAGCAGATATGGAAGGTGCACTGGACATTCCAGCTACATCACACACCTTGACTGGTCCCC AGACAACAAGTATATAATGTCTAACTCGGGAGACTATGAAATATTGTACTGGGTTTCTCCGGCATCATGATTGTGTACCGCCGGAAGCACCAGGAGCTGCAAGCCATGCAGATGGAGCTGCAGAGCCCTGAGTACAAGCTGAGCAAGCTCCGCACCTCGACCATCATGACCGACTACAACCCCAACTACTGCTTTGCTGGCAAGACCTCCTCCATCAGTGACCTGAAGGAGGTGCCGCGGAAAAACATCACCCTCATTCGGGGTCTGGGCCATGGCGCCTTTGGGGAGGTGTATGAAGGCCAGGTGTCCGGAATGCCCAACGACCCAAGCCCCCTGCAAGTGGCTGTGAAGACGCTGCCTGAAGTGTGCTCTGAACAGGACGAACTGGATTTCCTCATGGAAGCCCTGATCATCAGCAAATTCAACCACCAGAACATTGTTCGCTGCATTGGGGTGAGCCTGCAATCCCTGCCCCGGTTCATCCTGCTGGAGCTCATGGCGGGGGGAGACCTCAAGTCCTTCCTCCGAGAGACCCGCCCTCGCCCGAGCCAGC。
3) designing and synthesizing an amplification primer: taking the fusion result sequence obtained in the step 2) as a sequence template, carrying out sequence design, synthesis and purification on upstream and downstream amplification primers;
TABLE 1 sequences of upstream and downstream primers and related information
The upstream and downstream primer sequence design needs to meet the following requirements: (1) the number of base sequences of the upstream primer at the 3 'end position before the position of the upstream primer is separated from the fracture fusion sites of the two genes is not less than 45bp, and the number of base sequences of the downstream primer at the 5' end position after the position of the downstream primer is separated from the fracture fusion sites of the two genes is not less than 45 bp; (2) the G/C content is 40-60%, the length of the primers is 18-35 bp, and the complementary pairing number between the upstream primers and the downstream primers is not higher than 6 bp; (3) NCBI (https:// www.ncbi.nlm.nih.gov /) is specific for the upstream and downstream primers.
4) Amplification and verification of a reaction system: the method comprises the following steps: 4.1 preparing negative and positive RNA samples, diluting the positive RNA sample into a positive RNA sample with low mutation frequency according to a multiple proportion, and using the negative sample as a diluent in the dilution process; 4.2 reverse transcription of RNA sample, using the kit to carry out reverse transcription of RNA into cDNA; 4.3 reaction system preparation and PCR amplification: preparing a reaction system containing the upstream and downstream amplification primers, carrying out PCR amplification, and collecting a fluorescence signal after each cycle is finished; 4.4 judging the result: and reading the Tm value according to the amplification and dissolution curve, and judging the detection result.
TABLE 2 reaction System
TABLE 3 running conditions for PCR amplification
FIG. 1 shows the amplification and lysis curve of the negative sample, FIG. 2 shows the amplification and lysis curve of the positive sample at 2-fold, FIG. 3 shows the amplification and lysis curve of the positive sample at 10-fold, and FIG. 4 shows the amplification and lysis curve of the positive sample at 10-fold. Therefore, the fluorescence quantitative PCR detection method provided by the invention has high sensitivity and good specificity.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Sequence listing
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Claims (5)
1. The fluorescent quantitative PCR detection method of fusion gene mutation is characterized in that: the method comprises the following steps:
1) searching fusion gene sites: searching fusion gene information, searching exon fracture points, and searching transcript sequences from the exon fracture points to termination points;
2) fusion mutation sequences were analyzed in combination: merging the two sections of transcript sequences obtained in the step 1) to be used as a complete fusion result sequence;
3) designing and synthesizing an amplification primer: taking the fusion result sequence obtained in the step 2) as a sequence template, carrying out sequence design, synthesis and purification on upstream and downstream amplification primers;
4) amplification and verification of a reaction system: the method comprises the following steps: 4.1 preparing negative and positive RNA samples, diluting the positive RNA sample into a positive RNA sample with low mutation frequency according to a multiple proportion, and using the negative sample as a diluent in the dilution process; 4.2 reverse transcription of RNA sample, using the kit to carry out reverse transcription of RNA into cDNA; 4.3 reaction system preparation and PCR amplification: preparing a reaction system containing the upstream and downstream amplification primers, carrying out PCR amplification, and collecting a fluorescence signal after each cycle is finished; 4.4 judging the result: and reading the Tm value according to the amplification and dissolution curve, and judging the detection result.
2. The method for fluorescent quantitative PCR detection of fusion gene mutation according to claim 1, wherein: the fusion gene is EML4-ALK, and the exon fracture point comprises an EML4 gene exon20 fracture point and an ALK gene exon20 fracture point.
3. The method for fluorescent quantitative PCR detection of fusion gene mutation according to claim 2, characterized in that: the upstream and downstream amplification primers comprise: upstream primer TGGTCCCCAGACAACAAGTAT, SEQ ID NO: 1, downstream primer TTGGGGTTGTAGTCGGTCAT, SEQ ID NO: 2.
4. the method for fluorescent quantitative PCR detection of fusion gene mutation according to claim 2, characterized in that: the reaction system comprises the upstream and downstream amplification primers, amplification reaction liquid, cDNA and nuclease-free water; the PCR amplification operating conditions include: 30s at 95 ℃; 10s at 95 ℃, 30s at 60 ℃ and 40 cycles.
5. The method for fluorescent quantitative PCR detection of a mutation in a fusion gene according to claim 1 or 2, wherein: the kit is a cDNA one-chain synthesis kit.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719525A (en) * | 2012-04-12 | 2012-10-10 | 厦门艾德生物医药科技有限公司 | Primer, probe and detection kit for detection of EML4-ALK fusion gene mutation |
| CN103468813A (en) * | 2013-09-17 | 2013-12-25 | 广州达健生物科技有限公司 | EML4-ALK (Echinoderm microtubule associated protein like4-anaplastic lymphoma kinase) fusion gene fluorescent quantitative PCR (polymerase chain reaction) assay kit |
| CN103805684A (en) * | 2012-11-09 | 2014-05-21 | 益善生物技术股份有限公司 | PCR primer, kit and liquid phase chip for EML4-ALK fusion gene detection |
| CN105229175A (en) * | 2013-03-15 | 2016-01-06 | 雅培分子公司 | For increasing and measuring the method for RNA fusion gene variant, the method distinguishing them and relevant primer, probe and test kit |
| CN106978497A (en) * | 2017-04-26 | 2017-07-25 | 武汉友芝友医疗科技股份有限公司 | Detection primer, probe and the detection kit of EML4 ALK fusion gene mutations |
| CN109439751A (en) * | 2018-11-13 | 2019-03-08 | 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 kit of KIAA1549-BRAF fusion |
| CN109706232A (en) * | 2019-03-04 | 2019-05-03 | 合肥欧创基因生物科技有限公司 | For detecting primer, probe and the kit and its detection method of the fusion mutation of mankind's ALK gene |
-
2021
- 2021-06-09 CN CN202110642037.4A patent/CN113136419A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719525A (en) * | 2012-04-12 | 2012-10-10 | 厦门艾德生物医药科技有限公司 | Primer, probe and detection kit for detection of EML4-ALK fusion gene mutation |
| CN103805684A (en) * | 2012-11-09 | 2014-05-21 | 益善生物技术股份有限公司 | PCR primer, kit and liquid phase chip for EML4-ALK fusion gene detection |
| CN105229175A (en) * | 2013-03-15 | 2016-01-06 | 雅培分子公司 | For increasing and measuring the method for RNA fusion gene variant, the method distinguishing them and relevant primer, probe and test kit |
| CN103468813A (en) * | 2013-09-17 | 2013-12-25 | 广州达健生物科技有限公司 | EML4-ALK (Echinoderm microtubule associated protein like4-anaplastic lymphoma kinase) fusion gene fluorescent quantitative PCR (polymerase chain reaction) assay kit |
| CN106978497A (en) * | 2017-04-26 | 2017-07-25 | 武汉友芝友医疗科技股份有限公司 | Detection primer, probe and the detection kit of EML4 ALK fusion gene mutations |
| CN109439751A (en) * | 2018-11-13 | 2019-03-08 | 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 kit of KIAA1549-BRAF fusion |
| CN109706232A (en) * | 2019-03-04 | 2019-05-03 | 合肥欧创基因生物科技有限公司 | For detecting primer, probe and the kit and its detection method of the fusion mutation of mankind's ALK gene |
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