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Primer, probe and detection kit for detection of EML4-ALK fusion gene mutation

A detection kit and kit technology, applied in the biological field, can solve the problems of poor detection specificity, complicated operation, and long detection time of FISH method, and achieve the effect of wide clinical application range, wide detection range and fast detection speed

Active Publication Date: 2012-10-10
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0006] At present, the detection method of EML4-ALK fusion mutation is mainly the fluorescence in situ hybridization (FISH) method, however, the FISH method has poor detection specificity, low sensitivity, long detection time, and cannot detect multiple fusions produced by EML4-ALK fusion at the same time. variant
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 high-sensitivity detection method that can detect multiple mutations of the EML4-ALK fusion gene at the same time, so as to realize the detection of EML4-ALK with a rapid detection method. Simultaneous detection of multiple fusion mutations to provide scientific reference for clinical individualized treatment of lung cancer

Method used

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  • Primer, probe and detection kit for detection of EML4-ALK fusion gene mutation
  • Primer, probe and detection kit for detection of EML4-ALK fusion gene mutation
  • Primer, probe and detection kit for detection of EML4-ALK fusion gene mutation

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Effect test

Embodiment 1

[0080] Utilize the system of the present invention to detect plasmid, experimental plasmid template (containing EML4-ALK variant 1 mutation), utilize above-mentioned fluorescent PCR to detect the method for EML4-ALK gene fusion mutation as follows:

[0081] (1) Plasmid treatment and extraction:

[0082] 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 with Tris-HCl (10mmol / L, PH8.0) solution To 2ng / μL as a PCR template, take 5μL for PCR reaction amplification.

[0083] (2) Establish PCR amplification reaction system:

[0084] The cDNA template obtained above was used as a template for real-time fluorescent PCR amplification, and...

Embodiment 2

[0093] Using the present invention to detect clinical samples, 110 cases of paraffin-embedded tissue samples of clinical lung cancer to be tested were taken and sent to our company, including 65 cases of males and 45 cases of females, aged 34-75 years, with an average age of 54 years and a median age of 50 years . Utilize specific primer of the present invention and probe fluorescent PCR system to detect the gene fusion mutation step of the EML4-ALK of 110 clinical samples as follows:

[0094] (1) Sample processing and RNA extraction:

[0095] (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 precipitate, shake and mix (remove xylene), and centrifuge at room temperature at 14000RPM Centrifuge for 2 minutes to discard the supernatant, open the cap of the centrifuge tube, and dry at 37°C;

[0096] (b) Add 150 μl Buffer PKD and 10 μl protei...

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Abstract

The invention discloses a primer, a probe and a detection kit for detection of an EML4-ALK fusion gene mutation. The primer and probe provided by the invention are SEQ ID NO 1-SEQ ID NO 24. The primer and probe of the invention can carry out specific detection on EML4-ALK fusion gene mutation. The present invention has the following advantages: (1) an established real-time fluorescent PCR system capable of simultaneously detecting 9 kinds of fusion mutation of EML4-ALK gene; (2) high sensitivity capable of detecting mutation of 5-10 copies; (3) simple operation, cheap detection and wide clinical application range; (4) a wide range of samples from fresh pathological tissue to paraffin embedded tissue or pleural fluid; and (5) high speed detection with a detection procedure taking only 90 minutes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a detection kit for fusion gene 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, 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: small cell carcinoma (SCLC) and non-small cell lung cancer (NSCLC) in te...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 郑立谋张海龙施伟杰罗捷敏阮力周细武
Owner AMOY DIAGNOSTICS CO LTD
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