Fluorescent quantitative PCR (Polymerase Chain Reaction) method for detecting ALK (Anaplastic Lymphoma Kinase) fused gene and detection kit
A detection kit and gene fusion technology, applied in the field of molecular biology, can solve problems such as polluting the operating environment, complicated technology, false positives, etc., and achieve the effects of high sensitivity, simple detection operation, and strong specificity
Inactive Publication Date: 2014-10-01
NINGBO UNIV
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However, this technique has obvious defects: (1) it can only detect known mutations and cannot be used to detect new fusion mutations, so it will lead to false negatives; (2) simultaneous detection of multiple fusion types requires a large number of specimens, The technology is complex; (3) The subsequent sequencing steps after amplification are prone to the diffusion of amplicons and pollute the operating environment, resulting in false positives
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[0030] Example: Detection of ALK fusion gene expression
[0031] 400 non-small cell lung cancer samples (including tumor FFPE tissue, fresh tumor tissue, sputum, pleural effusion, and peripheral blood, etc.) of different clinical stages were collected. The total RNA of samples was extracted using the cell tissue RNA extraction kit and paraffin-embedded tissue (FFPE) total RNA extraction kit of Ningbo Youcheng Biomedical Technology Co., Ltd. The extracted RNA was dissolved in DEPC-treated water and stored in a -80°C refrigerator.
[0032] Design primers and fluorescent probes that can specifically detect the basic expression level (non-fusion expression, B) and overall expression level (non-fusion expression + fusion expression, T) of ALK gene, in which the basic expression level (B) area fluorescent probe is used HEX signal, total expression level (T) region fluorescent probe using FAM signal. The sequence is as follows:
[0033] ALK non-kinase region primer sequence:
[0...
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The invention relates to the field of molecular biology and specially relates to a method of detecting gene mutation by adopting fluorescent quantitative PCR (Polymerase Chain Reaction) and particularly relates to an ALK(Anaplastic Lymphoma Kinase) fused gene fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit with unlimited fusing types and a detection method. The method provided by the invention comprises the following steps: designing specific primers and probes in a kinase domain and a non-kinase domain; designing a fluorescent quantitative PCR reaction system, and amplifying the gene target sequence in a sample by using the specific primers; and detecting FAM and HEX fluorescent signal strengths in the reaction system by means of hybridization of the probes and the amplification product as a judgement standard of a result. The kit provided by the invention can detect ALK fused gene mutation with unlimited fusing types, is strong in specificity and high in sensitivity, can detect mutation as low as 10 copies, is fast in detection speed and can complete detection within 90 minutes.
Description
technical field [0001] The present invention relates to the field of molecular biology, in particular to a method for detecting fusion genes by fluorescent quantitative PCR, in particular to a fluorescent quantitative PCR detection kit for detecting anaplastic lymphoma kinase (Anaplastic Lymphoma Kinase, ALK) fusion genes And the detection method can detect as low as 10 copies of fusion gene mutation molecules in clinical samples with only 0.1% mutation content. Background technique [0002] Lung cancer is a malignant tumor with the highest morbidity and mortality worldwide. As a new and effective means of treating lung cancer, molecular targeted drugs have been more and more widely used in clinic. The targeted drug crizotinib, which specifically inhibits anaplastic lymphoma kinase (ALK), has a good curative effect on patients with non-small cell lung cancer with excessive ALK kinase activity due to ALK gene fusion. It was approved in 2011 Approved by the US Food and Drug ...
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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2563/107
Inventor 周细武黄清邱英华
Owner NINGBO UNIV