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Detection primer for EML4-ALK (anaplastic lymphoma kinase) fused gene mutation, probe and detection kit

A kit and probe technology, which is applied in the fields of biotechnology and medicine, can solve the problems of long time and failure to meet clinical needs, and achieve the effects of low production cost, accurate and reliable test results, and fewer raw material components

Inactive Publication Date: 2017-07-25
WUHAN YZY MEDICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Over time, new fusion mutation types of EML4 and ALK genes were gradually discovered, and the nine EML4-ALK fusion mutations detected by CN102719525A could no longer meet clinical needs
In addition, in the disclosed method, fluorescent PCR detection takes 90 minutes, reverse transcription PCR needs another 65 minutes, and the whole step takes a long time
At the same time, in order to ensure the accuracy of the clinical laboratory test results, it is best to add an anti-pollution system to the product, and simultaneously introduce a blank control to monitor the test reagent and the environment to prevent false positive results. This aspect is not mentioned in CN102719525A. and

Method used

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  • Detection primer for EML4-ALK (anaplastic lymphoma kinase) fused gene mutation, probe and detection kit
  • Detection primer for EML4-ALK (anaplastic lymphoma kinase) fused gene mutation, probe and detection kit
  • Detection primer for EML4-ALK (anaplastic lymphoma kinase) fused gene mutation, probe and detection kit

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Embodiment 1

[0044] The embodiment of the present invention is achieved in this way, a kit for EML4-ALK fusion gene detection, the kit includes PCR buffer, 13 sets of specific primers, 1 specific probe and quality control system, HotStart Taq Enzyme, UNG Enzyme.

[0045] The PCR buffer solution contains 1.0-5.0 mM MgCl2, 1.0-5.0 mM dNTPs, namely 1.0-5.0 mM each of dATP, dUTP, dGTP and dCTP.

[0046] The forward primers of the 13 groups of specific primer sequences are SEQ ID NO: 1-13, and the reverse primer sequence is SEQ ID NO: 14. Among them, SEQ ID NO: 1-13 are ARMS primers, which can specifically recognize 13 corresponding EML4-ALK fusion mutation types, and wherein SEQ ID NO: 14 is a common PCR primer. The 13 ARMS primers are paired with the bases to be amplified at the 3' template bases, and at the same time, one or two base mismatches are added at the penultimate 2-3 positions of the 3' end to enhance specificity.

[0047] The primer sequences are listed below:

[0048] EML4-ALK...

Embodiment 2

[0112] The EML4-ALK fusion gene detection kit prepared in Example 1 was used to detect the side sample.

[0113] In this embodiment, FFPE samples of 300 lung cancer patients were collected, RNA was extracted therefrom, and after reverse transcription into cDNA, the EML4-ALK fusion gene detection kit obtained in Example 1 was used to detect the EML4-ALK fusion mutation of the sample to be tested .

[0114] 1. RNA extraction from FFPE samples

[0115] (a) Take the above-mentioned lung cancer samples, add 1ml of xylene respectively, mix well, centrifuge at 13000rpm for 2 minutes at room temperature, discard the supernatant, add 1ml of absolute ethanol to the precipitate, shake and mix (remove xylene), and Centrifuge at 13000rpm for 2 minutes, discard the supernatant, open the cap of the centrifuge tube, and place it at 37°C for 10 minutes to evaporate the residual ethanol;

[0116] (b) Add 240 μL Buffer PKD and 10 μL proteinase K to the centrifuge tube, shake and mix, incubate ...

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Abstract

The invention relates to the fields of biotechnologies and medicines, particularly discloses a detection primer for EML4-ALK (anaplastic lymphoma kinase) fused gene mutation, a probe and a detection kit, and aims to detect the EML4-ALK fused gene mutation. The detection primer comprises sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 14. The sequence of the probe is as shown in SEQ ID NO: 15, of which the end 5' is modified with FAM or VIC (vasoactive intestinal contractor), and the end 3' is modified with NFQ-MGB (non-fluorescent quencher-myohemoglobin). The kit comprises the detection primer, the probe and a quality control system. The detection primer can specifically identify 13 EML4-ALK fusion mutation types, is high in sensitivity and can detect 5-copied mutations; the whole reverse transcription PCR (polymerase chain reaction) and fluorescence PCR detection process can be completed only by 80 minutes.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to the detection of EML4-ALK fusion gene mutation. Background technique [0002] Lung cancer is one of the most common malignant tumors in my country, and its 5-year survival rate is only 16.8%. According to the World Health Organization (WHO) cancer Globocan database, in 2012, there were 652,800 new lung cancer patients in my country, ranking first among all malignant tumors and accounting for about 1 / 3 of the world. [0003] Based on histopathological results, lung cancer can be divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), of which 80% are NSCLC. Most (>70%) lung cancer patients are diagnosed at an advanced stage, and systemic chemotherapy is the main treatment option. In recent years, with the development of molecular medicine and the continuous emergence of targeted drugs, the treatment of non-small cell lung cancer has entere...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6848C12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/156C12Q2600/166C12Q2531/113C12Q2561/101C12Q2521/531C12Q2545/113
Inventor 蔡从利张喆李丽琼周鹏飞
Owner WUHAN YZY MEDICAL SCI & TECH
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