Specific primer probe composition, kit and method for detecting T790M site of EGFR gene

A primer probe and kit technology, applied in the field of molecular biology, can solve problems such as unsuitable blood circulation free nucleic acid, and achieve the effects of short cycle, improved sensitivity and strong performance

Inactive Publication Date: 2018-04-03
中源协和基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The mutation of blood circulating free nucleic acid is often lower than the minimum detection limit of PCR or NGS, and the techniques based on these two schemes are not suitable for the detection of blood circulating free nucleic acid

Method used

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  • Specific primer probe composition, kit and method for detecting T790M site of EGFR gene
  • Specific primer probe composition, kit and method for detecting T790M site of EGFR gene
  • Specific primer probe composition, kit and method for detecting T790M site of EGFR gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1 detects the composition of the kit of human EGFR gene T790M mutation

[0058] A test kit for detecting the T790M 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 TE buffer solution and has a composition as 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 with the detection T790M mutation with the sequence shown in SEQ ID NO:3 and the detection with the sequence shown in SEQ ID NO:4 Wild-type fluorescent probe. The 5' end of the fluorescent probe for detecting T790M mutation is connected with a fluorescent reporter group, which is VIC; the 3' end of the fluorescent probe for detecting T790M mutation is connected with a quencher group, which is MGB; the 5' end of the fluorescent probe for detecting wild type A fluorescent reporter group is connected, ...

Embodiment 2

[0060] Embodiment 2 detects the method for human EGFR gene T790M mutation

[0061] 1. Using the kit described in Example 1;

[0062] 2. Prepare the PCR reaction system according to the following table:

[0063] 25μl Master Mix for 2×dd PCR, 9μl primer-probe mixture, 15μl template, 1μl Stabilize, 50μl in total; Explanation: The corresponding mixture preparation process has been described above.

[0064] 3. According to the operating instructions of the Raindrop Source chip, add the mixed solution of the PCR reaction system prepared in step 2 to the Raindrop Source chip, and prepare a new 8-tube tube and place it at the indicated position of the instrument;

[0065]4. After the microdroplets are generated, cover the 8-tube tubes containing the microdroplets with latex caps and place them in a 96-well PCR machine for amplification;

[0066] 95°C, 10min; another 40 cycles, each cycle including 95°C for 15sec, then 60°C for 60sec;

[0067] After 40 cycles, proceed at 98°C for 10...

Embodiment 3

[0070] Embodiment 3 uses the method in embodiment 2 to detect standard substance

[0071] The detection results of the standards with mutation ratios of 0.1%, 0.01% and 0.001% are as follows: Figure 1-Figure 3 As shown, the detection results of the wild-type standard were as follows Figure 4 As shown, wherein: the standard substance containing the mutation is obtained by the following method: EGFRT790M mutant genomic DNA and wild-type genomic DNA are synthesized and plasmids are constructed, and then according to the mutant type: the wild-type ratio is 1:1000, 1:10000, Mix at a ratio of 1:100000, and then break the mixed plasmid into a fragmented DNA of about 170bp that is close to the length of the ctDNA fragment; the negative quality control product is prepared by the following method: the DNA plasmid of the EGFR T790M wild type is broken, and the size is Fragmented DNA of about 170 bp with a similar length to the ctDNA fragment.

[0072] The standard substances whose mu...

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Abstract

The invention discloses a specific primer probe composition, a kit and a method for detecting a T790M site of an EGFR gene. The kit comprises an upstream primer, a downstream primer, a fluorescent probe I for detecting T790M mutation and a fluorescent probe II for detecting a wild type. For the kit, the primer pair with the specific sequences and the probes with the specific sequences are designedfor the human EGFR gene T790M mutation and reaction systems are optimized. According to the invention, through a method of a Raindropdigital PCR platform, high sensitivity detection on the T790M mutation of the EGFR gene is achieved and the abundance of the mutation is obtained simultaneously; the detection limit can be low to one in 100000. Furthermore, the invention can be used for detecting multi-source samples, comprising tumor tissue samples and ctDNAs, so that the application scope of the kit, reaction systems and method are expanded, and a medication guidance for treatment of patientswith lung cancer T790M mutation is provided.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a specific primer probe composition, kit and detection method for detecting the T790M site of EGFR gene based on Raindrop droplet digital PCR. 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 about 1.61 million new cases of lung cancer worldwide, and about 1.38 million deaths, ranking first among malignant tumors. Among them, non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) accounts for more than 80% of lung cancers. [0003] With the rapid development of translational medicine, the treatment of patients with advanced non-small cell lung cancer has shifted from the era of chemotherapy to the era of molecular targets, and the epidermal growth factor receptor (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/156C12Q2563/159C12Q2561/101
Inventor 李同恩罗玺师鸿翔陶然董炳楠陈兴京朱兵
Owner 中源协和基因科技有限公司
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