Primer, probe and detection reagent kit for detecting RET fusion gene

A detection kit and fusion gene technology, which are applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of poor specificity of FISH detection, affecting detection accuracy, and high reagent cost

Inactive Publication Date: 2015-07-01
张道允 +1
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the detection method of RET 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 the practical application of detection. It is urgent to develop a highly sensitive detection method that can simultaneously detect multiple RET gene fusions, so as to quickly detect multiple RET fusion mutations at the same time. To provide scientific reference for clinical individualized treatment of lung cancer

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  • Primer, probe and detection reagent kit for detecting RET fusion gene
  • Primer, probe and detection reagent kit for detecting RET fusion gene
  • Primer, probe and detection reagent kit for detecting RET fusion gene

Examples

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Embodiment 1

[0184] Using the system of the present invention to detect plasmids, plasmid templates for experiments (containing KIF5B-15 and RET-12 gene fusions), and using the above fluorescent PCR to detect RET gene fusion mutations are as follows:

[0185] (1) Plasmid treatment and extraction

[0186] The plasmid was extracted using the plasmid extraction kit of TIANGEN (HighPure Plasmid kid, DP116), and the specific extraction steps were performed according to the instructions of the kit. The extracted DNA was dissolved in Tris-HCl (10mM, pH 8.0), and the quality of the extraction was detected by a UV spectrophotometer to determine its concentration, and then the DNA concentration was adjusted to 20ng / ul with Tris-HCl (10mM, pH 8.0) solution as a diluted mother solution .

[0187] According to formula C 拷贝浓度 =(C 质量浓度 X6.02X10 23 ) / MW DNA Dilute wild-type plasmid to 10 6 copies / microliter.

[0188] (2) Establish PCR amplification reaction system:

[0189] The cDNA template obtai...

Embodiment 2

[0212] Using the present invention to detect clinical samples, 130 clinical lung cancer paraffin-embedded tissue samples to be tested were taken and sent, including 81 males and 49 females, aged 35-76 years, with an average age of 55 years and a median age of 51 years. Utilize specific primer of the present invention and probe fluorescent PCR system to detect the gene fusion mutation step of the RET of 130 cases of clinical samples as follows:

[0213] (1) Sample processing and RNA extraction

[0214]a. Take about 1×1 cm2 slices of each lung cancer sample (about 4-5 slices in total) and place them in a centrifuge tube (prepared by yourself), add 1 ml of xylene, cap the tube tightly, and vortex for 10 seconds.

[0215] b. Centrifuge at 12,000 rpm (~13,400×g) for 2 minutes, carefully aspirate and discard the supernatant, and be careful not to aspirate and discard the sediment. c. Add 1 ml of absolute ethanol and vortex to mix. Centrifuge at 12,000 rpm for 2 minutes, discar...

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Abstract

The invention discloses a primer and a probe for detecting an RET fusion gene. The primer comprises an RET fusion mutation specificity reverse transcription primer, an RET gene fusion mutation detection specificity primer and a probe nucleotide sequence. The invention discloses a detection reagent kit for detecting the RET fusion gene. The detection reagent kit comprises an mRNA reverse transcription cDNA system, wherein the mRNA reverse transcription cDNA system is used for detecting the PCR reaction of the detection reagent. The specific primer and probe technology is adopted, so that the human RET gene fusion mutation can be specifically detected; a real-time fluorescent PCR system is established, and 17 fusion mutations of the RET genes can be simultaneously detected; the sensitivity is high, the mutation of 5 to 10 copies can be detected; the operation is simple, the detection is cheap, and the clinical application range is wide; the specimen detection range is wide, and the specimen can be fresh pathological tissues, paraffin-embedded tissues or pleural fluid; the detection speed is high, and the detection process can be completed in 90 minutes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer, a probe and a detection kit for RET fusion gene detection. Background technique [0002] Lung cancer is the most common malignant tumor worldwide, and its morbidity and mortality rank first among all cancers. Every year, more than 1.3 million patients die of lung cancer worldwide, nearly half of which occur in developing countries. According to the statistics of the Ministry of Health of my country in 2010, the mortality rate of lung cancer was 30.83 / 100,000, and lung cancer has become the malignant tumor with the highest morbidity and mortality. (Orras JM, Fernandez E, Gonzalez JR, et al. Lung cancer mortality in European regions (1955-1997). Ann Oncol, 2003, 14(1): 159-161; Ministry of Health of the People's Republic of China, "2010 China Health Statistical Yearbook). [0003] Lung cancer can be divided into two categories based on histopathology: small cell lung can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/156C12Q2531/113C12Q2561/101C12Q2561/113
Inventor 张道允巩子英
Owner 张道允
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