Probe composition for detecting lung cancer mutant genes based on NGS method and kit

The technology of a kit and composition is applied in the field of lung cancer mutation gene detection composition to achieve the effect of ensuring the accuracy of results, saving time, and uniform coverage.

Pending Publication Date: 2020-02-14
基恩生物科技(大连)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, how to meet the detection of multiple types of samples at the same time, that is, to achieve multiple detections in one box, and to achieve early detection of lung cancer, is the current challenge for lung cancer gene detection kits

Method used

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  • Probe composition for detecting lung cancer mutant genes based on NGS method and kit
  • Probe composition for detecting lung cancer mutant genes based on NGS method and kit
  • Probe composition for detecting lung cancer mutant genes based on NGS method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The verification method adopted in the library building hybridization capture process of the present invention is as follows:

[0024] 1. Extract genomic gDNA or blood free ctDNA, both of which come from hospital clinical samples, use Qubit for quantitative and quality inspection

[0025] 2. Interrupt, end repair, add A

[0026] [Note: Perform a operation for tissue gDNA samples; perform b operation for liquid biopsy ctDNA samples]

[0027] 1. Set the following programs on the PCR instrument: the temperature of the hot cover is set to 70°C, and the volume of the reaction solution is set to 50 μL.

[0028] a: The gDNA reaction program is: 1 min at 4°C, 12 min at 32°C, 30 min at 65°C, and hold at 4°C.

[0029] b: ctDNA reaction program: 1 min at 4°C, 30 min at 20°C, 30 min at 65°C, hold at 4°C. Run the program and stop the program when the temperature of the PCR machine reaches 4°C.

[0030] 2. Place the thick-walled PCR tube on ice, add 200ng of gDNA or 25ng of ctDNA...

Embodiment 2

[0100] Embodiment 2 positive standard substance detection:

[0101] 1. Use international common standard products: Horizon (HD827) company and Coriell Institute (NA12878human reference gDNA) for detection.

[0102] 2. Qubit quantification, negative standard NA12878 and positive standard HD827 each 200ng to build a library.

[0103] 3. Perform library construction and sequencing according to the method described in Example 1 of the present invention, and use the Illumina platform for sequencing in Example 2.

[0104] 4. The analysis results are as follows:

[0105] (1) Test results of the organization version:

[0106] Gene mutation type Ref Alt mutation frequency NRAS SNPs G T 1.58％ DDR2 SNPs C G 2.46％ ALK SNPs A G 2.56％ PIK3CA SNPs G A 3.01％ PIK3CA SNPs G A 3.09％ PIK3CA SNPs A G 1.64％ ROS1 SNPs T C 4.17％ ROS1 SNPs C T 2.45％ EGFR SNPs T G 2.71％ MET SNPs G A 2....

Embodiment 3

[0111] Example 3 Tissue Detection

[0112]Clinical samples—an FFPE sample from a lung cancer patient was used for NGS database construction; according to the method described in Example 1, the panel composed of SEQ ID NO 1 to 75 probe sequences of the present invention was used for tissue plate detection:

[0113] 1. Extract genomic DNA from FFPE tissue, and use a total of 200ng of gDNA.

[0114] 2. Library construction was performed on the extracted gDNA, and sequencing was performed using the PE150 illumina sequencing platform.

[0115] 3. The analysis results are as follows:

[0116]

[0117]

[0118] A total of 8 mutation sites were detected in this sample, among which the EGFR p.E746_A750del mutation was a deletion mutation of exon 19, and the mutation frequency was 35.63%. Combined with the clinical diagnosis of the patient, the possibility of non-small cell lung cancer and adenocarcinoma. Therefore, the medication guidelines given for reference are: gefitinib, e...

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Abstract

The invention discloses a probe composition for detecting lung cancer gene mutation based on an NGS method and a kit thereof. The probe composition is selected from at least one of probes with nucleotide sequences as shown in SEQ ID NO.1-75, the kit is suitable for lung cancer gene mutation detection of FFPE, tissue and peripheral blood ctDNA based on the NGS method, and then the purposes of earlyscreening of lung cancer mutant genes, real-time monitoring of recurrence and the like are achieved. The uniquely designed UMI bimolecular tag can effectively reduce background noise, eradicate tracepollution, remove false positive and ensure the accuracy of a result, so that the sensitivity in ctDNA detection reaches 0.1%. A universal Short-Y joint is used in tissue detection, and the detectionsensitivity can reach 2%. The more possibilities are provided for accurate targeted therapy of patients. Tissue samples and plasma samples are similar in library building workflow, the simplicity ofthe workflow is guaranteed, time is saved, the efficiency is high, and the operation is easy.

Description

technical field [0001] The invention relates to a lung cancer mutation gene detection composition, in particular to a set of probe compositions and a kit for detecting lung cancer genes based on an NGS method. Background technique [0002] NGS has significant advantages in clinical tumor gene detection. Next-generation sequencing can detect common and rare mutations, gene fusion, gene amplification and other gene mutations at the same time, guide targeted drug use, and reveal drug resistance mechanisms. At the same time, NGS-based liquid biopsy technology can realize dynamic monitoring and prompt tumor recurrence and metastasis earlier. [0003] The free DNA in the blood is called cfDNA (Cell free DNA), and the somatic DNA of apoptotic cells or shed cancer cells is called ctDNA. ctDNA has the same genetic mutations or characteristics as the primary cancer cell DNA. This makes ctDNA extremely specific and can be used as a highly sensitive biomarker. There are many limitat...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6827C12Q1/6886
CPCC12Q1/6827C12Q1/6886C12Q2600/156C12Q2535/122C12Q2537/165
Inventor 丁岩
Owner 基恩生物科技(大连)有限公司
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