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Primer for detecting EML4-ALK fusion gene based on vesicles and kit

A technology of EML4-ALK-V2F and EML4-ALK-V1F, which is applied in the field of primers and kits for non-invasive detection of EML4-ALK fusion gene based on vesicles, can solve problems such as inability to provide detection, achieve convenient sampling, reduce pain, Sensitive effect

Inactive Publication Date: 2018-07-24
北京恩泽康泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

None of these methods provide effective detection in some patients in whom biopsies are difficult to obtain

Method used

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  • Primer for detecting EML4-ALK fusion gene based on vesicles and kit
  • Primer for detecting EML4-ALK fusion gene based on vesicles and kit
  • Primer for detecting EML4-ALK fusion gene based on vesicles and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 detects the plasmid containing the EML4-ALK-V1 fusion gene fragment

[0053] The plasmid DNA template used in the experiment is pTOPO-EML4-ALK-V1 (pTOPO is the name of the cloned plasmid). The specific method for detecting the mutation of the EML4-ALK-V1 fusion gene by real-time fluorescent quantitative PCR technology is as follows:

[0054] 1. The synthesis and real-time fluorescent quantitative PCR detection method of the plasmid containing the EML4-ALK-V1 fusion gene fragment

[0055] (1) Using the whole gene synthesis method, synthesize the EML4-ALK-V1 fusion gene fragment, which contains 200 bp of the EML4 gene fragment upstream of the EML4-ALK-V1 fusion site, and 200 bp of the ALK gene fragment downstream of the fusion site.

[0056] (2) Cloning the synthesized gene fragment into the pTOPO cloning vector to form the pTOPO-EML4-ALK-V1 cloning plasmid, transforming Escherichia coli, extracting the pTOPO-EML4-ALK-V1 plasmid, and performing a generation of s...

Embodiment 2

[0072] Example 2 detects the plasmid containing the EML4-ALK-V2 fusion gene fragment

[0073] The plasmid DNA template used in the experiment is pTOPO-EML4-ALK-V2 (pTOPO is the name of the cloned plasmid). The specific method for detecting the mutation of the EML4-ALK-V2 fusion gene by real-time fluorescent quantitative PCR technology is as follows:

[0074] 1. The synthesis and real-time fluorescent quantitative PCR detection method of the plasmid containing the EML4-ALK-V2 fusion gene fragment

[0075] (1) Using the whole gene synthesis method, synthesize the EML4-ALK-V2 fusion gene fragment, which contains 200 bp of the EML4 gene fragment upstream of the EML4-ALK-V2 fusion site, and 200 bp of the ALK gene fragment downstream of the fusion site.

[0076] (2) Cloning the synthesized gene fragment into the pTOPO cloning vector to form the pTOPO-EML4-ALK-V2 cloning plasmid, transforming Escherichia coli, extracting the pTOPO-EML4-ALK-V2 plasmid, and performing a generation of s...

Embodiment 3

[0092] Example 3 detects the plasmid containing the EML4-ALK-V3 fusion gene fragment

[0093] The plasmid DNA template used in the experiment is pTOPO-EML4-ALK-V3b (pTOPO is the name of the cloned plasmid). The specific method for detecting the mutation of the EML4-ALK-V3b fusion gene by real-time fluorescent quantitative PCR technology is as follows:

[0094] 1. The synthesis and real-time fluorescent quantitative PCR detection method of the plasmid containing the EML4-ALK-V3b fusion gene fragment (1) The method of whole gene synthesis is adopted to synthesize the EML4-ALK-V3b fusion gene fragment, which contains the EML4-ALK-V3b fusion The EML4 gene fragment upstream of the site is 200 bp, and the ALK gene fragment downstream of the fusion site is 200 bp.

[0095] (2) Cloning the synthesized gene fragment into the pTOPO cloning vector to form the pTOPO-EML4-ALK-V3b cloning plasmid, transforming Escherichia coli, extracting the pTOPO-EML4-ALK-V3b plasmid, and performing a gen...

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Abstract

The invention provides a set of primer for noninvasively detecting EML4-ALK fusion gene based on vesicles. The invention also provides a probe and a primer for detecting the EML4-ALK fusion gene through qPCR or digital PCR; the sequence is shown as SEQ ID NO:1-5. The primer and the probe are used for building a noninvasive, fast and high-sensitivity EML4-ALK fusion gene mutation detection method based on vesicles. The detection method has the advantages that by aiming at body fluid specimens (including blood, hydrothorax, ascitic fluid, cerebrospinal fluid and the like), clinical specimens canbe easily obtained. The detection sensitivity of the method is high; EML4-ALK mutation containing 10 copies can be stably detected; the precise medicine use guide can be provided.

Description

technical field [0001] The invention relates to molecular biology detection technology, in particular to a primer and a kit for non-invasive detection of EML4-ALK fusion gene based on vesicles. Background technique [0002] According to the "2017 China Tumor Registration Annual Report", the incidence and mortality of lung cancer in my country rank first among all malignant tumors. Among newly diagnosed patients, the proportion of patients with stage I lung cancer is only 15-20%. Most patients do not have the conditions for surgery, and drug therapy is still the main treatment for lung cancer. In recent years, molecular targeted drugs are gradually replacing traditional chemotherapy drugs as the first choice for the treatment of lung cancer due to their high specificity and low toxicity. Taking non-small cell lung cancer (NSCLC, accounting for about 85% of the total number of lung cancers) as an example, according to epidemiological research results, about 3-5% of NSCLC pati...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/156C12Q2563/159C12Q2561/113C12Q2563/107
Inventor 孔关义李好勋赵立波
Owner 北京恩泽康泰生物科技有限公司
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