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Detection kit and detection method for human BRAF gene V600E mutation

A detection kit and detection method technology, applied in the field of molecular biology, can solve problems such as difficult detection of ctDNA, and achieve the effects of saving experimental consumption and labor costs, saving detection costs, and simple and accurate interpretation

Inactive Publication Date: 2020-04-07
重庆浦洛通基因医学研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to solve the problem that ctDNA is difficult to detect in the peripheral blood of cancer patients with BRAF gene V600E mutation without obvious clinical manifestations or in the early stage of cancer

Method used

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  • Detection kit and detection method for human BRAF gene V600E mutation
  • Detection kit and detection method for human BRAF gene V600E mutation
  • Detection kit and detection method for human BRAF gene V600E mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] 1. Preparation of primers, probes and inhibitors included in the kit for detecting BRAF gene V600E mutation in human blood samples

[0053] According to the wild-type sequence of the BRAF gene (Gene ID: 673) published in the NCBI database, and taking the V600E mutation site (rs113488022) of the BRAF gene as a reference, specific arms-PCR primers and probes were designed. Using the mutant plasmid and wild-type plasmid constructed by genetic engineering as templates, a real-time fluorescent PCR detection system was established to achieve high sensitivity and high specificity detection of BRAF gene V600E mutation.

[0054] The primers and probes for detecting BRAF gene V600E mutation are characterized in that the detection primers and probes are designed according to the BRAF gene V600E mutation site, and the sequence is as follows:

[0055] Upstream primer: GTAAAAATAGGTGCTTTTGGTATAGCTGCCGA

[0056] Downstream primer: CTTTCTAGTAACTCAGCAGCATCTCAGG

[0057] Fluorescent pro...

Embodiment 2

[0110] The usage of the kit of the present invention will be described in detail below in conjunction with specific example 2. This example is implemented under the technical premise of the present invention, and the detailed implementation and specific operation process are given. In Example 2, the peripheral blood samples and puncture tissue samples of 10 patients with thyroid cancer were collected at the same time, and they were respectively named as No. 1-10 samples. The ctDNA in No. 1-10 peripheral blood was detected by the method of the present invention. The detection results of ctDNA in No. 1 peripheral blood were compared with the results of the first-generation sequencing of BRAF gene in No. 1-10 punctured tissue DNA to confirm the accuracy of the present invention.

[0111] 1. Detection method:

[0112](1) ctDNA extraction of clinical patient samples: ctDNA was extracted from the peripheral blood samples of thyroid cancer patients No. In the present invention, HiPu...

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Abstract

The invention provides a detection kit for human BRAF gene V600E mutation. The detection kit includes primers and a probe for detecting BRAF gene V600E mutation, an inhibitor, internal control primersand a probe, external control primers and a probe, and is characterized in that the detection primers are SEQ ID NO.1 and SEQ ID NO.2, the detection probe is SEQ ID NO.3; the inhibitor is SEQ ID NO.4; the internal control primers are SEQ ID NO. 5 and SEQ ID NO. 6, and the internal control fluorescent probe is SEQ ID NO. 7; and the external control primers are SEQ ID NO. 8 and SEQ ID NO. 9, and the external control fluorescent probe is SEQ ID NO. 10. According to the invention, the peripheral blood of patients is used to efficiently and noninvasively detect the mutation of BRAF gene in ctDNA.The detection kit has advantages of high detection sensitivity, short cycle, low cost, simple interpretation, and has great clinical promotion and application value.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a kit and a detection method for BRAF gene V600E mutation in peripheral blood circulating tumor DNA (ctDNA) of tumor patients. Background technique [0002] BRAF is a guanylate-binding protein RAS-activated serine / threonine protein kinase, which plays an important role in regulating the mitogen-activated protein kinase (MAPK) signaling pathway and is one of the most important proto-oncogenes. The MAPK signaling pathway can normally regulate cell growth, division and differentiation, and can also cause cancer due to the formation of oncogenic mutants of RAF family members. The vast majority of BRAF mutations occur in metastatic melanoma, colon, lung, and thyroid cancers. Among them, the production of BRAF (V600E) mutant significantly enhanced the activity of BRAF, which led to the uncontrolled division of cancer cells, and about 8% of human tumors had BRAF mutation. This mutatio...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858
CPCC12Q1/6858C12Q1/6886C12Q2600/156C12Q2600/166C12Q2531/113C12Q2545/101C12Q2561/101C12Q2535/137
Inventor 罗锋韩勋领孙松松
Owner 重庆浦洛通基因医学研究院有限公司
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