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Primer and probe composition, kit and detection method for detecting EGFR (epidermal growth factor receptor) gene mutation through multiple fluorescence pcr

A technology of multiple fluorescence and composition, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of high cost and relatively high sample size requirements, and achieve high accuracy and good specificity , high flux effect

Pending Publication Date: 2022-06-07
江苏吉诺思美精准医学科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, most of the EGFR detection kits on the market are single-fluorescence mutation detection, with a total of 7 holes, which require relatively high sample volume and high cost; among the multi-gene detection kits for lung cancer of Xiamen Aide Biomedical Technology Co., Ltd. EGFR mutation detection uses 3 fluorescence, a total of 4 wells

Method used

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  • Primer and probe composition, kit and detection method for detecting EGFR (epidermal growth factor receptor) gene mutation through multiple fluorescence pcr
  • Primer and probe composition, kit and detection method for detecting EGFR (epidermal growth factor receptor) gene mutation through multiple fluorescence pcr
  • Primer and probe composition, kit and detection method for detecting EGFR (epidermal growth factor receptor) gene mutation through multiple fluorescence pcr

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Embodiment 1

[0060] In this embodiment, the genetically engineered plasmid is used as the positive plasmid, and the negative clinical tissue sample is used as the negative control. The method for detecting 32 kinds of gene mutations of EGFR by multiple fluorescent PCR of the present invention comprises the steps:

[0061] (1) Clinical tissue sample preparation

[0062] Select 8 cases of negative clinical tissue samples that have been tested and dilute them to about 5ng / ul with TE buffer. The ddPCR quantification is about 1500copy / ul, and the sample volume is 2ul, which is about 3000copy.

[0063] (2) Plasmid dilution

[0064] The 32 kinds of EGFR positive plasmids synthesized were diluted 10 times with TE buffer to 30copy / ul, and the loading volume was 1ul, that is, 30copy positive plasmids.

[0065] (3) Construction of multiple fluorescent PCR amplification reaction system

[0066] 19del&S768I system

[0067]

[0068] L858R&EGFR 20ins system

[0069]

[0070]

[0071] T790M&...

Embodiment 2

[0085] A total of 55 cases of EGFR mutations in clinical tissue samples have been detected by using the present invention, and compared with digital PCR detection. Utilize specific primer of the present invention and probe detection step as follows:

[0086] (1) Prepare clinical tissue samples tested by digital PCR, including 25 negative samples and 30 positive samples, including 11 cases of 19del, 5 cases of L858R, 5 cases of T790M, 2 cases of S768I, 2 cases of EGFR 20ins, 2 cases of L861Q, and 3 cases of G719X For example, the samples are diluted to about 5ng / ul.

[0087] (2) Set up PCR amplification system: same as Example 1

[0088] The PCR reaction conditions are

[0089] The first stage: 1min at 95°C, 1 cycle

[0090] The second stage: 95°C for 15s, 64°C for 30s, 10 cycles

[0091] The third stage: 95°C for 15s, 60°C for 30s, 38 cycles

[0092] Signal collection: Collect FAM / VIC / ROX / CY5 signals at 60°C in the third stage, perform real-time PCR, and save the file.

...

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Abstract

The invention discloses a primer and probe composition for detecting EGFR (epidermal growth factor receptor) gene mutation through multiple fluorescent PCR (polymerase chain reaction), a kit and a detection method, the primer and probe composition comprises an EGFR gene mutation detection specific primer pair, an EGFR gene specific probe, an amplification blocking probe and an internal control system, and the sequences of the primer and probe composition are shown as SEQ ID NO 1 to SEQ ID NO 44. 32 mutations of the EGFR gene can be simultaneously detected in three reaction tubes, the detection sensitivity is 1%, the operation is simple, the cost is saved, the sample size requirement is low, and the clinical application range is wide. The kit can be used for detecting fresh tissue samples, FFPE samples and hydrothorax and ascites samples.

Description

technical field [0001] The invention belongs to the technical field of EGFR gene mutation detection, and in particular relates to a primer and probe composition, a kit and a detection method for multiplex fluorescent PCR detection of EGFR gene mutation. Background technique [0002] Lung cancer is currently the largest cancer in the world, and the incidence and mortality of lung cancer rank first among all tumors. Among lung cancer patients, non-small cell lung cancer (NSCLC) accounts for about 80-85%. Currently, targeted therapy has become an important means of clinical treatment for non-small cell lung cancer (NSCLC). EGFR is a transmembrane receptor tyrosine kinase. The activation of this region, that is, phosphorylation, is of great significance to the signal transmission related to the proliferation and growth of cancer cells. Therefore, EGFR, as a molecular target of NSCLC, has received widespread attention and has been developed successively. Many clinical trials hav...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6886C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/156C12Q2600/166C12Q2531/113C12Q2537/143C12Q2545/113C12Q2563/107C12Q2535/137
Inventor 高如霞孙克京洪兵邢红兵
Owner 江苏吉诺思美精准医学科技有限公司
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