EML4-ALK (Echinoderm microtubule associated protein like4-anaplastic lymphoma kinase) fusion gene fluorescent quantitative PCR (polymerase chain reaction) assay kit

A detection kit and fluorescence quantitative technology, applied in the field of molecular biology, can solve the problems of cumbersome steps, time-consuming, and difficulty in popularization and promotion.

Active Publication Date: 2013-12-25
广州达健生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method involves two rounds of PCR and sequencing, the step

Method used

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  • EML4-ALK (Echinoderm microtubule associated protein like4-anaplastic lymphoma kinase) fusion gene fluorescent quantitative PCR (polymerase chain reaction) assay kit
  • EML4-ALK (Echinoderm microtubule associated protein like4-anaplastic lymphoma kinase) fusion gene fluorescent quantitative PCR (polymerase chain reaction) assay kit
  • EML4-ALK (Echinoderm microtubule associated protein like4-anaplastic lymphoma kinase) fusion gene fluorescent quantitative PCR (polymerase chain reaction) assay kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Example 1: Detection of EML4-ALK subtype 1 (E13; A20) fusion gene A pair of probes and primers designed to specifically detect the EML4-ALK subtype 1 (E13; A20) fusion gene:

[0109] Variant 1 - forward primer: 5'-GAGTCATGCTTATATGGAGCAAAACT-3' (SEQ ID NO.1);

[0110] Variant 1 - reverse primer: 5'-TCAGCTTGTACTCAGGGCTCTG-3' (SEQ ID NO.2);

[0111] Variant 1-Taqman-probe: 5'-FAM-CCTAAAGTGTACCGCCGGAAGCACC-TAMRA-3' (SEQ ID NO.3);

[0112] Then optimize the reaction system for FQ-PCR detection: 15 μl reaction system, 0.15 μl (20 μM) of forward and reverse primers, 0.2 μl (20 μM) of each probe, 2.5 μl of sample template DNA (100-300 ng / μl), 2 *Taqman universal PCR.

[0113] Master Mix (purchased from Applied Bio, USA) 7.5 μl, ddH 2 O4.3 μl.

[0114] PCR reaction conditions: pre-denaturation at 95°C for 30 sec; and 65 cycles of amplification reaction at 95°C for 5 seconds and 58°C for 33 seconds. At the same time, in the fluorescence quantitative test, while testing the s...

Embodiment 2

[0115] Example 2: Detection of EML4-ALK subtype 2 (E20; A20) fusion gene

[0116] Design a pair of probes and primers that can specifically detect the EML4-ALK subtype 2 (E20; A20) fusion gene:

[0117] Variant 2 - forward primer: 5'-AAGTATATAATGTCTAACTCGGGAGACTATGA-3' (SEQ ID NO.4);

[0118] Variant 2 - reverse primer: 5'-AGCAGTAGTTGGGGTTGTAGTCG-3' (SEQ ID NO.5);

[0119] Variant 2-Taqman-probe: 5'-FAM-TTGTACTTGTACCGCCGGAAGCACCA-TAMRA-3' (SEQ ID NO.6);

[0120] Then optimize the reaction system for FQ-PCR detection: 15 μl reaction system, 0.15 μl (20 μM) of forward and reverse primers, 0.2 μl (20 μM) of each probe, 2.5 μl of sample template DNA (100-300 ng / μl), 2 *Taqman universalPCR

[0121]Master Mix (purchased from Applied Bio, USA) 7.5 μl, ddH 2 O4.3 μl.

[0122] PCR reaction conditions: pre-denaturation at 95°C for 30 sec; and 65 cycles of amplification reaction at 95°C for 5 seconds and 58°C for 33 seconds. At the same time, in the fluorescence quantitative test, ...

Embodiment 3

[0123] Example 3: Detection of EML4-ALK subtype 3a (E6a; A20) fusion gene

[0124] Design a pair of probes and primers that can specifically detect EML4-ALK subtype 3a (E6a; A20) fusion gene:

[0125] Variant 3a - forward primer: 5'-ACCAAAACTGCAGACAAGCATAAAG-3' (SEQ ID NO. 7);

[0126] Variant 3a - reverse primer: 5'-AGCTTGCTCAGCTTGTACTCAGG-3' (SEQ ID NO. 8);

[0127] Variant 3a-Taqman-probe: 5'-FAM-TGTCATCATCAACCAAGTGTACCGCCG-TAMRA-3' (SEQ ID NO.9);

[0128] Then optimize the reaction system for FQ-PCR detection: 15 μl reaction system, 0.15 μl (20 μM) of forward and reverse primers, 0.2 μl (20 μM) of each probe, 2.5 μl of sample template DNA (100-300 ng / μl), 2 *Taqman universal PCR

[0129] Master Mix (purchased from Applied Bio, USA) 7.5 μl, ddH 2 O4.3 μl.

[0130] PCR reaction conditions: pre-denaturation at 95°C for 30 sec; and 65 cycles of amplification reaction at 95°C for 5 seconds and 58°C for 33 seconds. At the same time, in the fluorescence quantitative test, w...

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Abstract

The invention relates to an EML4-ALK (echinoderm microtubule associated protein like4-anaplastic lymphoma kinase) fusion gene fluorescent quantitative PCR (polymerase chain reaction) assay kit, which is applicable to assay of EML4-ALK fusion gene mutation in lung adenocarcinoma. The kit comprises probes, primers and positive controls, which are specially designed for conserved sequences of 9 fusion variations of the EML4-ALK fusion gene. The kit can be used for quickly and accurately assaying 9 most common EML4-ALK fusion gene variations with high sensitivity, namely 9 fusion variations of 7 variant subtypes, which are subtype 1 (E13; A20), subtype 2 (E20; A20), subtype 3 (E6a/b; A20), subtype 4 (E14; A20), subtype 5 (E2a/b; A20), subtype 6 (E18; A20) and subtype 7 (E14; A20), so that a real-time fluorescent quantitative PCR system for assaying 9 most common EML4-ALK fusion gene variations can be established.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a kit for detecting EML4-ALK fusion gene fluorescence quantitative PCR, which is suitable for detection of EML4-ALK fusion gene mutation in lung adenocarcinoma. Background technique [0002] Lung cancer is a malignant tumor with the highest morbidity and mortality in the world. Over the past 50 years, the incidence and mortality of lung cancer have risen rapidly in countries all over the world, especially in industrialized countries. In 1995, 600,000 people died of lung cancer worldwide, and the number is increasing every year. In 2003, the World Health Organization (WHO) announced that the mortality rate was 1.1 million per year, and the incidence rate was 1.2 million per year. The cause of lung cancer is still not completely clear. Medical statistics show that the main cause of lung cancer is smoking (including second-hand smoke). 80% of male lung cancer and 75% of female lung c...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 邵琦
Owner 广州达健生物科技有限公司
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