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Kit for quantitatively detecting ALK, RET and ROS1 fusion genes based on ddPCR

A fusion gene, CCDC6-RET technology, applied in the field of nucleic acid in vitro diagnostic reagents, to achieve the effect of good applicability and high sensitivity

Inactive Publication Date: 2021-05-28
PILLAR BIOSCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, there are currently no products on the market that use ddPCR technology to detect EML4-ALK, CCDC6-RET and CD74-ROS1 fusion genes

Method used

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  • Kit for quantitatively detecting ALK, RET and ROS1 fusion genes based on ddPCR
  • Kit for quantitatively detecting ALK, RET and ROS1 fusion genes based on ddPCR
  • Kit for quantitatively detecting ALK, RET and ROS1 fusion genes based on ddPCR

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1 (taking the ddPCR operation of BIO-RAD company as an example)

[0052] 1. RNA preparation:

[0053] RNA was extracted from fresh tissue or FFPE, and RNA concentration was quantified using Qubit.

[0054] 2. Reverse transcription

[0055] RNA loading amount: 20-300ng / person

[0056] The reverse transcription system is as follows:

[0057] Reagent name Volume (μL) cDNA Master Mix 2 RNA 20-300ng nuclease-freewater Replenish water to 10ul total capacity 10ul

[0058] The amplification procedure is as follows:

[0059] temperature time 25℃ 10min 50℃ 10min 85℃ 5min 8℃ hold

[0060] 3. PCR reaction system preparation

[0061]

[0062]

[0063] 4. Microdroplet preparation

[0064] 4.1 Put a new DG8 cartridge into the holder;

[0065] 4.2 Add 20μl of the system to the 8 wells in the middle row of the DG8 cartridge; figure 1 shown.

[0066] 4.3 Add 70 μl of droplet gener...

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Abstract

The invention discloses a kit for quantitatively detecting ALK, RET and ROS1 fusion genes based on ddPCR. The kit comprises a negative reference substance, a positive reference substance, a PCR reaction solution I and a PCR reaction solution II. The invention provides an RT-PCR quantitative detection system based on ddPCR. The RT-PCR quantitative detection system comprises a forward primer, a reverse primer and a fluorescent probe, wherein the forward primer and the reverse primer aim at fusion genes EML4-ALK, CCDC6-RET and CD74-ROS1 and an internal control gene TBP. According to the kit, a forward primer is designed on a 5'partner gene, a probe and a reverse primer are designed on a 3'driver gene to detect ALK, RET and ROS1 fusion genes, and a detection result is expressed by a copy number in unit volume. Through adoption of a ddPCR detection method, whether EML4-ALK, CCDC6-RET and CD74-ROS1 fusion occurs in a clinical sample can be simply and rapidly detected, an important basis is provided for diagnosis, typing, clinical treatment and prognosis of ALK, RET and ROS1 related tumor diseases, and a new thought is provided for clinical treatment.

Description

technical field [0001] The invention belongs to the field of nucleic acid in vitro diagnostic reagents, in particular to a kit for quantitatively detecting ALK, RET and ROS1 fusion genes based on ddPCR. Background technique [0002] Tumor occurrence and development is a complex biological process involving multiple genes. With the in-depth study of tumor molecular pathology and molecular genetics, the medical community's understanding of tumors has changed from a disease in the traditional sense to a group of diseases driven by genes. Gene fusion is an important oncogene in solid and hematological malignancies. ALK, RET and ROS1 proto-oncogenes play a very important role in the occurrence of solid tumors. The detection of ALK, RET and ROS1 fusion genes brings new benefits to molecular precision therapy. new hope. [0003] The ALK gene encodes an intracellular tyrosine kinase signal receptor, which is located on the short arm of chromosome 2, contains 29 exons, and has a pr...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6851
CPCC12Q1/6886C12Q1/6851
Inventor 赵艳红王强
Owner PILLAR BIOSCI INC
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