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Primer pair, probe and kit for rapidly detecting ROS1 gene fusion mutation and use method thereof

A gene fusion and primer pair technology, applied in the field of molecular biology, can solve the problems of complex detection process and missed detection, and achieve the effect of simple experimental operation, avoiding false negatives, and high cost performance

Pending Publication Date: 2021-03-26
杭州求臻医学检验实验室有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the common methods of ROS1 gene fusion mainly include fluorescence in situ hybridization (FISH), immunohistochemistry (ICH), reverse transcription fluorescent PCR (RT-PCR) and next-generation sequencing (NGS). Missed detection, some methods have complicated detection process

Method used

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  • Primer pair, probe and kit for rapidly detecting ROS1 gene fusion mutation and use method thereof
  • Primer pair, probe and kit for rapidly detecting ROS1 gene fusion mutation and use method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Test sample processing and RNA extraction

[0029] 1) Test samples include fresh tumor tissue, frozen section tissue, paraffin-embedded tissue or section;

[0030] 2) For fresh tumor tissue or frozen section tissue, first disperse the cells, add 300 μL of lysate per 10-20 mg of tissue, and grind the tissue thoroughly with a grinding pestle; then add 590 μL of nuclease-free water and 10 μL of protease to the homogenate K, after mixing, treat at 56°C for 10-20min, then centrifuge to take the supernatant, and extract total RNA according to the operation of the RNA prep pure Tissue Kit (TIANGEN).

[0031] 3) For paraffin-embedded tissues or slices, take 2-8 slices with a thickness of 5-10 μM, quickly place them in a centrifuge tube with 1.5 mL of nuclease-free water, add 1 mL of xylene, vortex to mix, centrifuge and discard. Add 1mL of absolute ethanol to the supernatant, vortex to mix, discard the supernatant after centrifugation, and extract the total RNA according to th...

Embodiment 2

[0034] 4. Detection of ROS1 fusion genotype by micro-droplet digital PCR

[0035] 1) Micro-droplet digital PCR detection process of ROS1 fusion genotype

[0036] The micro-droplet digital PCR reaction conditions used for detection are as follows: a reverse transcription ddPCR (RT-ddPCR) amplification system was prepared, and the sequences of primers and probes used are shown in Table 1.

[0037] Table 1

[0038]

[0039] RT-ddPCR amplification reaction mixture includes: 1×reverse transcriptase mix (Bio-Rad), 2×Master Mix premix (Bio-Rad), 1×stabilizer (Bio-Rad), add primer probe mixture , respectively 200-1000nM (ROS1-F1, ROS1-R1, ROS1-F2, ROS1-R2), each detection probe 100-800nM (ROS1-Pb1, ROS1-Pb2). Add 1-100ng of template RNA, add water to 30μL, and mix the reagents evenly.

[0040] Put the PCR plate containing the prepared 20 μL digital PCR mixture, a new 96-well PCR plate, tip, droplet generation card and droplet generation oil into the AutoDG automatic droplet genera...

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Abstract

The invention relates to the technical field of digital PCR, and in particular relates to a primer pair, probe and kit for quickly detecting ROS1 gene fusion mutation and a use method thereof. A primer pair and probe for detecting ROS1 gene fusion mutation are included; the primer pair and the probe are designed according to a target area to be detected; according to the primer pair and the probe,a kit for rapidly detecting ROS1 gene fusion mutation is provided; the kit comprises the primer pair and the probe; and a use method of the primer pair, the probe or the kit is included. The method comprises the following steps of: S1, extracting to obtain a tumour tissue RNA sample; S2, performing digital PCR reaction on an RNA sample by using the primer pair, the probe or the kit; and S3, judging whether ROS1 gene fusion mutation occurs in the sample or not. The method disclosed by the invention is not affected by fusion partners and fusion sites; gene fusion causing ROS1 gene transcriptionabnormality can be accurately found; known and unknown fusion variations are detected at the same time; false negative is avoided; experimental operation is simple; the detection period is short; andthe cost performance is high.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a pair of primers, a probe, a kit and the use thereof for rapidly detecting ROS1 gene fusion mutation. Background technique [0002] The reactive oxygen species gene 1 (reactive oxygen species 1, ROS1) consists of a tyrosine kinase domain, a transmembrane domain and an extracellular domain containing an N-terminal glycosylation site, encoding a receptor tyrosine Kinase, in patients with non-small cell lung cancer (NSCLC), the positive rate of ROS1 rearrangement is about 1-2%, and the rearrangement site mainly occurs in exons 32-36 of the ROS1 gene. At present, it has been found that ROS1 can be fused with multiple genes, such as CD74, SLC34A2, EZR, LRIG3 and SDC4, etc. After fusion, it will continue to activate the ROS1 tyrosine kinase region and downstream PI3K / AKT, RAS / MAPK and other signaling pathways, thereby cause tumors. [0003] Clinical studies have shown that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6851C12Q2600/158C12Q2600/156C12Q2600/106C12Q2531/113C12Q2563/159C12Q2563/107
Inventor 段小红杨春燕张腾龙赵梓君承康平周启明
Owner 杭州求臻医学检验实验室有限公司
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