Detection kit for fusion mutation of ROS1 and various genes

A detection kit, fusion mutation technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. The effect of wide clinical application, wide detection range and fast detection speed

Inactive Publication Date: 2015-08-12
张道允 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection method of ROS1 fusion mutation is mainly the fluorescence in situ hybridization (FISH) method, but the detection specificity of the FISH method is poor, the sensitivity is low, and the detection time is long.
In addition, FISH detection requires expensive special instruments and equipment, the cost of reagents is high, and the operation is complicated, which limits the widespread use of clinical detection.
FISH testing requires that the test samples should not be stored for too long. Using samples with a long storage time for testing will lead to false negatives and affect the accuracy of the test.
Therefore, the FISH detection method cannot meet the needs of clinicians for practical application. It is urgent to develop a highly sensitive detection method that can detect multiple mutations of ROS1 fusion genes at the same time, so as to realize the rapid detection of multiple fusion mutations of ROS1. Simultaneous detection, so as to provide scientific reference for clinical individualized treatment of lung cancer

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  • Detection kit for fusion mutation of ROS1 and various genes
  • Detection kit for fusion mutation of ROS1 and various genes
  • Detection kit for fusion mutation of ROS1 and various genes

Examples

Experimental program
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Effect test

Embodiment 1

[0125] Using the system of the present invention to detect plasmids, the experimental plasmid template (containing CD47-E6 and ROS1-E32 gene fusion), and the method for detecting ROS1 gene fusion mutation using the above-mentioned fluorescent PCR are as follows:

[0126] (1) Plasmid processing and extraction

[0127] The plasmid was extracted using TIANGEN (HighPure Plasmid kid, DP116) plasmid extraction kit, and the specific extraction steps were performed according to the kit instructions. The extracted DNA is dissolved in Tris-HCl (10mM, pH 8.0), and the extraction quality is detected by UV spectrophotometer to determine its concentration, and then the DNA concentration is adjusted to 20ng / ul with Tris-HCl (10mM, pH 8.0) solution as the dilution mother liquid .

[0128] According to formula C Copy concentration =(C Concentration X6.02X10 23 ) / MW DNA Dilute the wild-type plasmid to 10 6 Copies / μl.

[0129] (2) Establish a PCR amplification reaction system:

[0130] Use the cDNA ...

Embodiment 2

[0147] Using the present invention to detect clinical samples, 115 cases of clinical lung cancer paraffin-embedded tissue samples to be tested by our company, 69 cases of males and 46 cases of females, aged 35-76 years old, average age 56 years old, median age 52 years old . The steps for detecting ROS1 gene fusion mutations of 115 clinical samples by using the specific primers and probe fluorescent PCR system of the present invention are as follows:

[0148] (1) Sample processing and RNA extraction

[0149] a. Take about 1×1cm2 of each lung cancer sample slice (about 4-5 slices in total) and place it in a centrifuge tube (self-prepared), add 1ml of xylene, cover the tube tightly, and vortex for 10 seconds.

[0150] b. Centrifuge at 12,000 rpm (~13,400×g) for 2 minutes, carefully aspirate and discard the supernatant, and be careful not to aspirate the sediment.

[0151] c. Add 1ml of absolute ethanol, vortex and mix well. Centrifuge at 12,000 rpm for 2 minutes, discard the supernata...

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Abstract

The invention discloses a detection kit for fusion mutation of ROS1 and various genes. The detection kit comprises a specific reverse transcription primer, a Q-PCR specific primer and a probe, wherein the nucleotide sequences of the Q-PCR specific prime and the probe comprise Mix 1: CD47; SDC4; SLC34A2 and ROS1 gene fusion, Mix 2: CD47; EZR; SDC4; SLC34A2 and ROS gene fusion, Mix 3: LRIG3; TPM3; GOPC and ROS1 gene fusion, Mix 4: GOPC and ROS1 gene fusion. By adopting a specific primer and probe technology, the detection kit disclosed by the invention can be used for specifically detecting human ROS1 gene fusion mutations. A real-time fluorescent PCR system is established for simultaneously detecting 15 fusion mutations of an RET gene; the detection kit is high in sensitivity, and 5-10-copy mutations can be detected; the detection kit is wide in sample detection range and high in detection speed.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a detection kit for the fusion mutation of ROS1 and multiple genes. Background technique [0002] Lung cancer is the main cause of cancer deaths in the world. It can be divided into two categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) from the histopathological point of view. Among them, non-small cell lung cancer accounts for approximately 85% of total lung cancer cases. In my country, the 5-year survival rate of lung cancer patients is only 13%, and about 70% of NSCLC are in the advanced stage at the time of diagnosis. The treatment effect and survival rate of small cell lung cancer (SCLC), which has a lower degree of differentiation and faster metastasis, is even less optimistic. The main reason is that most lung cancers lack high-sensitivity gene mutation detection and diagnosis technology, as well as matching scientific treatment plans, so th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 张道允巩子英
Owner 张道允
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