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Primer, probe and assay kit for detecting v-ros avian UR2 sarcoma viral oncogene homolog 1 (ROS1) gene fusion mutation

A detection kit, the technology of the kit, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problem of no ROS1 fusion gene mutation, unsatisfactory direct sequencing method, and low direct sequencing sensitivity and other problems, to achieve the effects of cheap detection, fast detection, and wide clinical application.

Active Publication Date: 2012-11-14
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sensitivity of direct sequencing is low, and the detection sensitivity is only 20-30%; the detection operation is complicated and the efficiency is low, and it usually takes 1-2 days to obtain the detection result
Therefore, the direct sequencing method cannot meet the actual needs of clinical detection, and there is an urgent need to develop a rapid fusion gene detection method in order to realize the simultaneous detection of fusion mutations using rapid detection methods
[0007] At present, there is no detection method and detection kit for ROS1 fusion gene mutation. The present invention develops a fast and easy-to-operate ROS1 fusion gene mutation detection kit and detection method for the first time.

Method used

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  • Primer, probe and assay kit for detecting v-ros avian UR2 sarcoma viral oncogene homolog 1 (ROS1) gene fusion mutation
  • Primer, probe and assay kit for detecting v-ros avian UR2 sarcoma viral oncogene homolog 1 (ROS1) gene fusion mutation
  • Primer, probe and assay kit for detecting v-ros avian UR2 sarcoma viral oncogene homolog 1 (ROS1) gene fusion mutation

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Effect test

Embodiment 1

[0064] Using the system of the present invention to detect plasmids, experimental plasmid templates (containing ROS1 fusion), the method of utilizing the above-mentioned fluorescent PCR to detect ROS1 gene fusion mutations is as follows:

[0065] (1) Plasmid treatment and extraction:

[0066] Plasmids were extracted using a plasmid extraction kit from TIANGEN (HighPure Plasmid Kit, DP116), and the specific extraction steps were performed according to the instructions of the kit. The extracted DNA was dissolved in Tris-HCl (10mmol / L, PH8.0), and the quality of the extraction was detected by a UV spectrophotometer to determine its concentration, and then the DNA concentration was adjusted to 2ng / μL was used as a PCR template, and 5 μL was used for PCR reaction amplification.

[0067] (2) Establish a PCR amplification reaction system:

[0068] Use the cDNA template obtained above as a template for real-time fluorescent PCR amplification, and perform PCR amplification according ...

Embodiment 2

[0078] Using the present invention to detect clinical paraffin-embedded tissue samples of lung cancer, 4 cases of ROS1 fusion positive and 11 cases of ROS1 wild-type samples determined by direct sequencing method were taken, and the ROS1 of 15 cases of clinical samples were detected by using the specific primer and probe fluorescent PCR system of the present invention The fusion gene mutation steps are as follows:

[0079] (1) Sample processing and RNA extraction:

[0080] (a) Take each of the above lung cancer samples, add 1ml of xylene, mix well, centrifuge at 14000RPM for 2min at room temperature, discard the supernatant, add 1ml of absolute ethanol to the pellet, shake and mix (remove xylene), 14000RPM at room temperature Centrifuge for 2 minutes to discard the supernatant, open the cap of the centrifuge tube, and dry at 37°C;

[0081] (b) Add 150 μl Buffer PKD and 10 μl proteinase K to the centrifuge tube, shake and mix, incubate at 56°C for 15 minutes, and incubate at 8...

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Abstract

The invention discloses a primer, a probe and an assay kit for detecting v-ros avian UR2 sarcoma viral oncogene homolog 1 (ROS1) gene fusion mutation, wherein the primer and the probe comprises the following sequences of SEQ ID NO 1 to SEQ ID NO 8. A specific primer and probe technique is adopted, and can specifically detect the human ROS1 gene fusion mutation. The method has the advantages that (1) a real-time fluorescent polymerase chain reaction (PCR) system is built to simultaneously detect four types of ROS1 gene fusion mutation; (2) the sensitivity is high, the mutation of 10-20kb can be detected; (3) the method is simple to operate and low in detection, and has a wide clinical application scope; (4) the sample detection scope is wide, and the sample can be fresh pathological tissues and paraffine-embedded tissues; and (5) the detection speed is fast, and the detection process can be completed within 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 detecting ROS1 gene fusion mutation. 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. The mortality rate of lung cancer in my country is 30.83 / 100,000, and lung cancer has become the first malignant tumor in terms of morbidity and mortality in our country. (Orras JM, Fernandez E, Gonzalez JR, et al. Lung cancer mortality in European regions (1955-1997). Ann Oncol, 2003, 14(1): 159). [0003] The 5-year survival rate of lung cancer patients in developed countries can reach about 20%, while the 5-year survival rate of lung cancer patients in my country is only 13%. It makes patients unable to receive scientific treatment plan in time, and makes patients miss the best time...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 张海龙江风阁罗捷敏阮力郑立谋
Owner AMOY DIAGNOSTICS CO LTD
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