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A fast, probe-based technology, applied in the fields of cancer detection and molecular biology, can solve problems such as weak correlation, achieve the effects of avoiding pollution, accurate and reliable experimental results, and streamlined operations
Pending Publication Date: 2020-11-27
杭州布平生物医药科技有限公司
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Immunohistochemistry or Western Blot is an important way to detect the protein level expression of EGFRvIII, but many studies have shown that the correlation between EGFRvIII protein level expression and nucleic acid level is weak
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Embodiment 1
[0022] A method for rapid detection of EGFRvIII mutation, comprising the following steps:
[0023] (1) extract the RNA of the tumor tissue of sample 1;
[0024] (2) Construct PCR reaction system;
[0025] (3) Real-time fluorescent quantitative PCR was carried out by TaqMan probe method;
[0026] (4) Interpretation of results.
[0027] Further, the extraction steps for sample 1 RNA are as follows: paraffin sample dewaxing: take five 7 μm sample 1 paraffin tissue sections and put them into a 1.5ml centrifuge tube, add 1ml xylene, centrifuge at 13,000xg for 1 minute, discard the supernatant, and then Add 1ml of xylene, centrifuge at 13,000xg for 1 minute, and discard the supernatant. Add 1ml of absolute ethanol to the centrifuge tube, centrifuge at 13,000xg for 1 minute, discard the supernatant, and dry it; add tissue lysate and proteinase K to the dried tissue, lyse at 56°C for 15 minutes, and transfer the centrifuge tube to Keep it at 80°C for 15 minutes and take it off. Wh...
Embodiment 2
[0039] A method for rapid detection of EGFRvIII mutation, comprising the following steps:
[0040] (1) extract the RNA of sample 1 tumor tissue;
[0041] (2) construct PCR reaction system;
[0042] (3) Real-time fluorescent quantitative PCR was carried out by TaqMan probe method;
[0043] (4) Interpretation of results.
[0044] Further, the extraction steps for sample 1 RNA are as follows: paraffin sample dewaxing: take five 7 μm sample 1 paraffin tissue sections and put them into a 1.5ml centrifuge tube, add 1ml xylene, centrifuge at 13,000xg for 1 minute, discard the supernatant, and then Add 1ml of xylene, centrifuge at 13,000xg for 1 minute, and discard the supernatant. Add 1ml of absolute ethanol to the centrifuge tube, centrifuge at 13,000xg for 1 minute, discard the supernatant, and dry it; add tissue lysate and proteinase K to the dried tissue, lyse at 56°C for 15 minutes, and transfer the centrifuge tube to Keep it at 80°C for 15 minutes and take it off. When it r...
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Abstract
The invention discloses a method for rapidly detecting EGFRvIII mutation. EGFRvIII RNA is used as a starting template of a reaction, redundant intron sequences in an EGFRvIII DNA sequence are avoided,and a probe crosses a fusion site of an EGFRvIII exon, so that interference of genome DNA is effectively avoided, and a detection result is more accurate; reverse transcription and real-time PCR share one reaction system, which avoids pollution possibly caused by repeated cover opening compared with traditional detection that carries out amplification by using cDNA as a template after reverse transcription; by changing fluorophores, a housekeeping gene ACTB is introduced into a single system, so that the risk of false negative is reduced, and the risk of false positive is reduced by identifying the result of [delta]Ct; a designed PCR product is relatively small, so that a paraffin-embedded sample with a high degradation degree can meet detection requirements, and meanwhile, the detectionresult can be in correspondence with an EGFRvIII immunohistochemical result of the same tissue sample. The method is simple in operation, and guarantees credibility and accuracy of the detection result.
Description
technical field [0001] The invention relates to the fields of cancer detection and molecular biology, especially a method for rapidly detecting EGFRvIII mutation. Background technique [0002] Malignant tumors are currently one of the diseases that seriously affect human health. The internal cause of malignant tumors is that changes in genes in cells lead to abnormal regulation of cell growth, resulting in abnormal proliferation and tumor formation. Many studies have shown that the EGFR (Epidermal Growth Factor Receptor) protein, one of the members of the surface growth factor receptor family, is related to the regulation mechanism of inhibiting tumor cell proliferation, angiogenesis, tumor invasion, metastasis and apoptosis. By binding to ligands, EGFR receptors can activate downstream signaling channels to regulate physiological processes such as cell growth, proliferation, and differentiation. Therefore, excessive activation of EGFR signaling pathways will lead to uncontr...
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