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Probe pool for detecting NTRK-1-2-3 fusion gene variation based on NGS method and kit thereof

A technology of TP53-NTRK1 and BTBD1-NTRK3, which is applied in the field of probe pools for detecting mutations of NTRK-1-2-3 fusion genes, achieves the effects of reducing RNA usage, high specificity, and reducing sequencing costs

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

AI Technical Summary

Problems solved by technology

[0004] However, there is no NTRK-1-2-3 fusion gene detection kit on the domestic market at present, so there is an urgent need to develop an NTRK-1-2-3 detection kit to make up for the blank detection of NTRK-1-2-3 fusion gene The phenomenon

Method used

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  • Probe pool for detecting NTRK-1-2-3 fusion gene variation based on NGS method and kit thereof
  • Probe pool for detecting NTRK-1-2-3 fusion gene variation based on NGS method and kit thereof
  • Probe pool for detecting NTRK-1-2-3 fusion gene variation based on NGS method and kit thereof

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Experimental program
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Embodiment 1

[0018] The verification method adopted in the library building hybridization capture process of the present invention is as follows:

[0019] 1. Extract RNA from FFPE or fresh tissue collected in the hospital.

[0020] 1. The following operations need to be performed in an environment free of RNase and DNase.

[0021] 2. For quantification, the optimal range of RNA is between 40ng-500ng (total amount).

[0022] 3. Agilent Technologies 4200Bioanalyzer quality inspection RNA fragments are complete, the main peak is between 200bp-6000bp, and fragments can be detected at 18s and 28s.

[0023] 2. Synthesis of cDNA by reverse transcription of RNA.

[0024] 1. RNA Fragmentation

[0025] (1) Mix 5 μL RNA sample and 5 μL FBS1 evenly, and centrifuge briefly.

[0026] (2) Set the following program to perform the reaction on the PCR instrument, and set the temperature of the hot cover to 94°C.

[0027]

[0028] 2. Synthesis of first-strand cDNA

[0029] (1) Take out the fragmenta...

Embodiment 2

[0125] Embodiment 2 positive standard substance detection:

[0126] 1. Purchase international common standard (SeraCare: FFPE NTRK Fusion RNAReference Material) from SeraCare Company.

[0127] 2. Qubit quantification, NTRK-1-2-3 positive standard using 50ng reverse transcription.

[0128] 3. Using the probe pool whose nucleotide sequence is shown as SEQ ID NO 1-74, construct the library according to the method described in Example 1, perform sequencing, and use the Illumina platform for sequencing.

[0129] 4. The detection data of NTRK-1-2-3 positive standard products are shown in Table 1 below:

[0130] Table 1. NTRK-1-2-3 positive standard detection data

[0131] Fusion RNA breakpoint 1 breakpoint 2 Breakpoint 1 Depth Breakpoint 2 Depth frequency LMNA-NTRK1 chr1:156108398 chr1:156844697 1061 674 48％ IRF2BP2-NTRK1 chr1:234744192 chr1:156844362 457 4724 49％ SQSTM1-NTRK1 chr5:179252226 chr1:156844362 2635 305 29％ T...

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Abstract

The invention discloses a probe pool for detecting NTRK-1-2-3 fusion gene variation based on an NGS method and a kit thereof. The probe pool is selected from at least one of probes with nucleic acid sequences as shown in SEQ ID NO: 1-74, and the kit is suitable for detecting the NTRK-1-2-3 fusion gene variation of FFPE and fresh tissues based on an NGS method. The capture probe is designed according to the highly related region of the NTRK-1-2-3 fusion gene variation, the coverage rate is high and uniform, and the specificity of fusion detection is improved. The library conversion rate is high, the RNA usage amount is low, and the sequencing cost is low. According to the present invention, the hot spot targeting mutant gene is covered, the probe capture specificity is high, and in addition, one product can meet the detection requirements of a variety of pan-cancer NTRK-1-2-3 fusion genes, can solve the clinical sample preparation problem, and can reduce the complex problem of repeatedwork of experimental workers.

Description

technical field [0001] The present invention relates to the technical field of a probe pool for detecting mutations of NTRK-1-2-3 fusion genes, in particular to a probe pool and a kit for detecting mutations of NTRK-1-2-3 fusion genes based on an NGS method. Make up for the blank of NTRK fusion gene detection in the domestic market. Background technique [0002] High-throughput sequencing technology is marked by the ability to sequence hundreds of thousands to millions of DNA molecules in parallel at a time and generally having a short read length. The Illumina sequencing platform is currently the mainstream next-generation sequencing platform, which can simultaneously detect common and rare mutations, gene fusion, gene amplification and other gene mutations, guide targeted drug use, and reveal drug resistance mechanisms. [0003] NTRK-1-2-3 gene fusions are chromosomal alterations that result in conformationally activated abnormal TRK fusion proteins that function as tumor...

Claims

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Application Information

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