Kit, reaction system and method for detecting human EGFR gene mutation

A reaction system and kit technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as inability to obtain detection results, small ctDNA fragments, etc., and achieve flexible reaction throughput, increased sensitivity, and stable results Effect

Inactive Publication Date: 2017-02-22
GENETRON HEALTH (BEIJING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in ctDNA, the mutation frequency is often lower than 1%, and the ctDNA fragments are small, so ARMS-PCR cannot obtain good detection results

Method used

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  • Kit, reaction system and method for detecting human EGFR gene mutation
  • Kit, reaction system and method for detecting human EGFR gene mutation
  • Kit, reaction system and method for detecting human EGFR gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 detects the composition of the kit of human EGFR gene L858R mutation

[0039] A kit for detecting the L858R mutation of the human EGFR gene, comprising: a primer-probe mixed solution and a digital PCR reaction premix, wherein: the primer-probe mixed solution is dissolved in a TE buffer solution with the composition shown in SEQ ID NO: 1 The upstream primer of the sequence, the downstream primer with the sequence shown in SEQ ID NO: 2, the fluorescent probe for detecting the L858R mutation with the sequence shown in SEQ ID NO: 3 and the detection with the sequence shown in SEQ ID NO: 4 The wild-type fluorescent probe, wherein: the concentration of the upstream primer and the downstream primer in the primer-probe mixture is 12 μM, the detection of the fluorescent probe for the L858R mutation and the detection of the wild-type fluorescent probe in the primer-probe mixture The concentration is 3 μM, and the preparation method of the primer-probe mixture is as f...

Embodiment 2

[0041] Embodiment 2 detects the method for human EGFR gene L858R mutation

[0042] 1. Using the kit described in Example 1;

[0043] 2. Prepare the PCR reaction system according to the following table:

[0044]

[0045] 3. According to the instructions of CHIP-LOADER, add the mixed solution of the PCR reaction system prepared in step 2 to the digital PCR chip, cover the chip with mineral oil, seal the chip and check to ensure that there is no leakage;

[0046] 4. Put the chip into the dedicated PCR instrument and set it according to the following amplification procedures;

[0047]

[0048] 5. After the amplification is completed, take out the chip from the PCR instrument, place it at room temperature for 10 minutes, and add it to the chip scanner for reading;

[0049] 6. The computer calculates the mutation ratio according to the fluorescent signal.

Embodiment 3

[0050] Embodiment 3 uses the method in embodiment 2 to detect standard substance

[0051] The detection results of the standards with mutation ratios of 38%, 2% and 0.1% are as follows: Figure 1-Figure 3 As shown, the detection results of the wild-type standard were as follows Figure 4 shown, from image 3 It can be seen that the sensitivity of detecting the EGFR gene L858R mutation can reach 0.1% using the kit and detection method of the present application, wherein: the standard product is EGFR mutant cell line genomic DNA and wild-type cell line genome with a known mutation ratio of 75% DNA was obtained after mixing in different proportions.

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Abstract

The invention discloses a kit, reaction system and method for detecting human EGFR gene mutation. The kit comprises an upstream primer, a downstream primer, a fluorescent probe for detecting an L858R mutation and a fluorescent probe for detecting wild types. The primers with specific sequences and the probes with the specific sequences are designed in the kit according to the L858R mutation of an EGFR gene. In an optimized reaction system, high-sensitivity detection of the L858R mutation of the EGFR gene is achieved through a digital PCR platform. Besides, negativity and positivity of the L858R mutation are detected, meanwhile the proportion of the mutation in a sample can be obtained, sources of samples capable of being detected in the kit are further diversified, the samples can be tumor tissue and ctDNAs, and the application range of the kit, the reaction system and the method is widened.

Description

technical field [0001] The invention relates to a kit, a reaction system and a method for detecting gene mutations, in particular to a kit, a reaction system and a method for detecting human EGFR gene mutations. Background technique [0002] Lung cancer has always been the leading cause of cancer-related deaths worldwide. According to the cancer report released by the International Agency for Research on Cancer of the World Health Organization, there are estimated to be about 1.61 million new cases of lung cancer worldwide, and about 1.38 million deaths, accounting for 13% and 18% of new cases and deaths of malignant tumors, ranking first among malignant tumors. first. Among them, non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) accounts for more than 80% of lung cancer. Although the diagnostic methods and treatment methods of lung cancer are continuously improved, the mortality rate of lung cancer has not been effectively controlled, and the prognosis of pati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q1/6886C12Q2600/118C12Q2600/156C12Q2600/158C12Q2563/107
Inventor 阎海王思振焦宇辰郑延军徐大勇郑乔松
Owner GENETRON HEALTH (BEIJING) CO LTD
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