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Method for monitoring secondary drug resistance of lung cancer patient to tyrosine kinase inhibitor through ddPCR technology

A technology of tyrosine kinases and inhibitors, which is applied in the fields of medicine and biology, and can solve the problems of lack, low plasma free DNA content, difficult detection of free DNA types and sequences, etc.

Pending Publication Date: 2016-08-10
上海张江转化医学研发中心有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, due to the extremely small amount of plasma cell-free DNA in the blood and the interference of a large amount of blood-derived DNA, it is still difficult to accurately isolate and purify DNA with high-efficiency DNA enrichment methods and high-sensitivity detection methods (such as sequencing, digital PCR). and reliably detect the specific types and sequences of tumor-associated cell-free DNA
For example, it has been reported in the literature that although the average content of circulating cell-free DNA (4771ng / ml) in patients with colorectal cancer is significantly different from that in healthy people (0.85ng / ml), for 67 patients with carcinoembryonic antigen (CEA) Only 47% of the test results are meaningful for diagnosis
[0008] Therefore, although the detection technology based on free blood DNA is the focus of research and development, there is still a lack of satisfactory mutant DNA detection technology in this field that can truly meet the clinical needs of high sensitivity, high accuracy and high reliability.

Method used

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  • Method for monitoring secondary drug resistance of lung cancer patient to tyrosine kinase inhibitor through ddPCR technology
  • Method for monitoring secondary drug resistance of lung cancer patient to tyrosine kinase inhibitor through ddPCR technology
  • Method for monitoring secondary drug resistance of lung cancer patient to tyrosine kinase inhibitor through ddPCR technology

Examples

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Effect test

Embodiment 1

[0198] Example 1 Detection of EGFR gene mutations in serum / plasma by ddPCR technology to monitor lung cancer patients' secondary resistance to tyrosine kinase inhibitors

[0199] (1) Take 4 mL of peripheral blood from the patient, separate the plasma / serum, and use the "QIAamp Circulating Nucleic Acid Kit" to extract the free DNA in the plasma / serum. High-concentration and high-purity DNA was obtained through sample dissolution, enrichment, column elution, and finally AVE elution, and finally the fragment size (<313bp) was determined by agarose gel electrophoresis to ensure the extraction quality.

[0200] (2) Reaction system preparation

[0201] a. Design of probes and primers: The present invention adopts the conventional Taqman probe method to design, and respectively designs wild-type and mutant probes for the target fragment of the EGFR gene. The FAM fluorescent channel marks the mutant type, and the HEX fluorescent channel marks the wild-type. , the wild-type and mutant...

Embodiment 2

[0214] Example 2 Detection of EGFR gene mutations in serum / plasma by ddPCR technology to monitor secondary drug resistance of lung cancer patients to tyrosine kinase inhibitors

[0215] (1) Take 4 mL of peripheral blood from the patient, separate the plasma / serum, and use the "QIAamp Circulating Nucleic Acid Kit" to extract the free DNA in the plasma / serum. High-concentration and high-purity DNA was obtained through sample dissolution, enrichment, column elution, and finally AVE elution, and finally the fragment size (<313bp) was determined by agarose gel electrophoresis to ensure the extraction quality.

[0216] (2) Reaction system preparation

[0217] a. Design of probes and primers: The present invention adopts the conventional Taqman probe method to design, and respectively designs wild-type and mutant probes for the target fragment of the EGFR gene. The FAM fluorescent channel marks the mutant type, and the HEX fluorescent channel marks the wild-type. , the wild-type and...

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Abstract

The invention provides a method for monitoring secondary drug resistance of a lung cancer patient to a tyrosine kinase inhibitor through ddPCR technology, and an application of the method to monitoring and control of the tyrosine kinase inhibitor in a process of treating a lung cancer patient. The method comprises: firstly, extracting DNA from peripheral plasma of a lung cancer patient, then detecting a mutation condition of a drug-resistant mutation site T790M of EGFR in the DNA through adoption of ddPCR technology, and finally judging whether the patient produces drug resistance according to the mutation situation.

Description

technical field [0001] The invention relates to the fields of medicine and biotechnology, in particular to a molecular detection technology that can be used to guide tumor treatment, especially a method for highly sensitive detection of EGFR gene T790M site mutation in peripheral plasma / serum and its role in monitoring lung cancer patients' response to tyrosine Acid kinase inhibitors produce secondary drug resistance and other aspects of the application. Background technique [0002] With the development of biomedical technology, molecular targeted drugs have been widely used in the treatment of tumors. Compared with conventional chemotherapy, targeted therapy has the advantages of better effect and less side effects. Clinical application results show that some patients will develop drug resistance to targeted drugs. Research by the American Cancer Society shows that more than 90% of tumor patients are affected by drug resistance to varying degrees during treatment. The dr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 丁飞飞许骋吴云鸣易静张桢珍楼敬伟朱陵君王剑鹏方圆蒋晓东李军川
Owner 上海张江转化医学研发中心有限公司
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