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Kit and method for multiple detection of ROS1 fusion gene mutation

A multiplex detection and kit technology, applied in the fields of biotechnology and tumor diagnosis, can solve the problems of cumbersome operations, sequence splicing burden, and insufficient high-throughput sequencing methods.

Pending Publication Date: 2020-04-07
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

IHC has the advantages of simplicity, cheapness, and mature operation methods, but there are challenges in sensitivity and reproducibility
[0008] (4) High-throughput sequencing method: High-throughput sequencing method is expensive, and the read length is about 30-250bp, which imposes a burden on sequence splicing. The detection results still need to be verified, and it is very important for detecting the junction of exons and introns. Insufficient accuracy for small fragment deletions
The high-throughput sequencing method is cumbersome to operate, which limits the sequencing speed

Method used

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  • Kit and method for multiple detection of ROS1 fusion gene mutation
  • Kit and method for multiple detection of ROS1 fusion gene mutation
  • Kit and method for multiple detection of ROS1 fusion gene mutation

Examples

Experimental program
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Effect test

Embodiment 1

[0111] The invention provides a method, primers, probes and kits for multiple gene fusion detection of lung cancer. The specific implementation steps are as follows:

[0112] (1) Extraction of nucleic acid templates from samples to be tested

[0113] Sun Yat-sen University Daan Gene Co., Ltd. nucleic acid extraction or purification reagent (Yue Sui Ji Bei 20170666), nucleic acid extraction was performed according to the kit instructions. The nucleic acid after tissue sample extraction is diluted to a concentration of 2-100ng / μL, and the purity should meet A 260 / A 280 The ratio ranges between 1.7-2.3. The template can be used directly for subsequent experiments or stored at -80°C for future use, avoiding repeated freezing and thawing.

[0114] (2) Sample addition

[0115] Take 3 μL of the RNase system and add it to the PCR reaction tube. Centrifuge briefly for 15s.

[0116] Take 5 μL of the negative quality control product, the sample nucleic acid template prepared in s...

Embodiment 2

[0132] Embodiment 2 Sensitivity and accuracy detection

[0133] The sensitivity reference product with a total nucleic acid concentration of 100ng / μL is composed of the detection gene locus plasmid or cell line, and the nucleic acid of the wild-type cell line is mixed in a certain proportion, and the 100ng / μL mixture contains 10 copies / μL of the fusion gene.

[0134] (1) Sample addition

[0135] Take 3 μL of the RNase system and add it to the PCR reaction tube. Centrifuge briefly for 15s.

[0136] Take 5 μL negative quality control substance, sensitivity reference substance (100 ng / μL total nucleic acid contains 10 copies / μL fusion gene), positive quality control substance, and add them to the reaction tube in sequence according to Table 5. Close the cap of the PCR reaction tube tightly, mix well, and centrifuge briefly for 10s.

[0137] For each sensitivity reference product, 5 repeated experiments.

[0138] (2) Real-time fluorescent PCR amplification:

[0139] Set the r...

Embodiment 3

[0147] Embodiment 3 clinical sample detection

[0148] Extraction of test sample nucleic acid:

[0149] (1) Extraction of nucleic acid templates from clinical samples to be tested

[0150] Collect 100 cases of clinically negative samples, the known SLC34A2-ROS1 (SLC34A2 Exon4; ROS1 Exon32), SLC34A2-ROS1 (SLC34A2 Exon4; ROS1 Exon34), SDC4-ROS1 (SDC4 Exon2; ROS1 Exon32), EZR-ROS1 (EZR Exon10; ROS1 Exon34), CD74-ROS1 (CD74 Exon6; ROS1 Exon32), CD74-ROS1 (CD74 Exon6; ROS1 Exon34) mutation clinical samples were randomly mixed in, Sun Yat-sen University Daan Gene Co., Ltd. nucleic acid extraction or purification reagents (tissue samples You can use Yuesui Machinery No. 20170666) to extract nucleic acid according to the kit instructions. The nucleic acid after tissue sample extraction is diluted to a concentration of 2-100ng / μL, and the purity should meet A 260 / A 280 The ratio ranges between 1.7-2.3. The template can be used directly for subsequent experiments or stored at -80°C...

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Abstract

The invention provides a kit and method for multiple detection of ROS1 fusion gene mutation and particularly, discloses primers and probes for multiple detection of ROS1 fusion gene mutation, and a kit comprising a primer and probe mixed solution. Six kinds of ROS1 fusion gene mutation types can be detected at the same time. The kit and method for multiple detection of ROS1 fusion gene mutation have the following advantages: the detection process is simple, the multiple detectable mutation types can be detected, the sensitivity is high, and the sample dependence is low and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology and tumor diagnosis, in particular, the invention relates to a kit and a method for multiple detection of ROS1 gene mutation. Background technique [0002] Lung cancer is one of the malignant tumors with the highest morbidity and mortality, among which non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancer patients, and the 5-year survival rate of advanced non-small cell lung cancer patients is about 15%. With the development of technology, more and more attention has been paid to gene mutations in tumors. Non-small cell lung cancer has a high degree of heterogeneity, resulting in extremely diverse clinical manifestations and therapeutic effects. [0003] Reactive oxygen species gene 1 (reactive oxygen species 1, ROS1) fusion gene mutation is more common in non-small cell lung cancer (NSCLC), accounting for about 2.4% of lung adenocarcinoma cases. The ROS1 gene encodes a recepto...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143
Inventor 蒋析文邓洁朱小亚黄志文钟灵秀
Owner DAAN GENE CO LTD
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