EML4-ALK fusion-gene non-invasive detection kit

A technology that combines genes and kits, and is applied in recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc. It can solve problems such as difficulties and high costs, and achieve high detection sensitivity.

Inactive Publication Date: 2018-02-02
SHENZHEN HUADA GENE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Therefore, it is very difficult to detect all types of fusion genes through DNA, and the cost will be higher due to the use of more probes; if t...

Method used

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  • EML4-ALK fusion-gene non-invasive detection kit
  • EML4-ALK fusion-gene non-invasive detection kit
  • EML4-ALK fusion-gene non-invasive detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, be used for the design and screening of the primer of digital PCR detection EML4-ALK fusion gene

[0052] 1. Design of primers and probes

[0053] According to the EML4-ALK fusion gene variant1 / 2 / 3 three mutations (Table 2), the primers and probes for detection were designed and synthesized.

[0054] Table 2 is the type of fusion gene

[0055]

[0056] In the NCBI database, find the nucleotide sequence of each gene, and use the software oligo7 to design primers and probes. On the basis of meeting the important principles as much as possible, the amplification products of the upper and lower primers are required to be less than 200bp, and the annealing temperature of the probes is higher than that of the primers. 5°C, it is expected to design 5-6 sets of primers and 1 set of probes for each gene, and additionally design internal reference primers and internal reference probes (see Table 3 for details):

[0057] Table 3 is the designed primer sequence a...

Embodiment 2

[0076] Embodiment 2, the establishment of the method for detecting EML4-ALK fusion gene by digital PCR

[0077] 1. Exosomal RNA extraction

[0078] The sample to be tested is an EML4-ALK fusion gene mutation cell line (purchased from Nanjing Kebai Biotechnology Co., Ltd.; the EML4-ALK mutation frequency of the cell line itself has been proved by next-generation sequencing, and its mutation frequency is 45%). Reagent screening for exosomes, exosome enrichment and exosome RNA extraction is as follows:

[0079] 1. Exosome enrichment

[0080] Use ExoQuickTC Exosomes Precipitation Solution, EXOTC10A-1, systembioscience (SBI) kit for exosome enrichment:

[0081] 1) Centrifuge 10ML of cell culture medium (3000g, 15min, 4°C), and take the supernatant (precipitated as cells or cell debris);

[0082] 2) Transfer the supernatant to a sterile test tube, add 2ML of ExoQuick-TC, invert the test tube and mix well.

[0083] 3) Refrigerate at 4°C overnight (at least 12h) (the test tube sho...

Embodiment 3

[0153] Embodiment 3, digital PCR detection EML4-ALK fusion gene method

[0154] 1. Extraction of plasma exosomal RNA

[0155] Using the SBI method screened in Example 2 to enrich exosomes + QIAGEN to extract exosomal RNA to extract lung cancer patients with EML4-ALK fusion gene mutations (it has been confirmed by sequencing that they contain a fusion gene mutation of EML4(13)-ALK(20) ) of exosomal RNA.

[0156] 2. Digital PCR detection

[0157] According to the second method of Example 2, the plasma exosomal RNA was detected by digital PCR with the best primers and the best probes.

[0158] The microdroplets with VIC fluorescence signal and FAM fluorescence signal contain at least 3 kinds of mutations, and are positive, that is, mutant microdroplets; the stronger the VIC fluorescence signal, the higher the mutation frequency of positive samples; the microdroplets with only FAM fluorescence signal If it does not contain any of the three mutations and is negative, it is a wil...

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Abstract

The invention discloses an EML4-ALK fusion-gene non-invasive detection kit. A set of primers are provided and include EFTUD2-F1, EFTUD2-R1, EML4(13)-ALK-F2, EML4(20)-ALK-F3, EML4(6)-ALK-F4, EML4(6/13/20)-ALK-R, a detection probe ALK-EML4(6/13/20)-TAR, and a reference probe EFTUD2-REF. According to the EML4-ALK fusion-gene non-invasive detection kit, non-invasive plasma exosome is adopted to detectEML4-ALK fusion genes, the formula of an optimal RNA extraction agent is explored, the optimal RNA extraction agent serves as a template, and then digital PCR detection is conducted by the adoption of the found optimal primers and probes; the EML4-ALK fusion-gene non-invasive detection kit is high is detection sensibility, rapid, convenient and absolutely quantified, and guides drugs for the EML4-ALK fusion genes.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a non-invasive detection kit for EML4-ALK fusion gene. Background technique [0002] Lung cancer is one of the diseases with the highest morbidity and mortality in the world, among which non-small cell lung cancer accounts for about 80-85% of lung cancer. Currently, about 10 driver genes have been identified in lung adenocarcinoma. The clinical use of molecular targeted therapy drugs targeting these driving genes has made significant progress in the treatment of lung cancer and the prolongation of survival. Therefore, finding more new therapeutic targets has become a hot spot in lung cancer research today. In addition to well-known gene mutations such as EGFR and KRAS, the EML4-ALK fusion gene has also been confirmed as a therapeutic target for non-small cell lung cancer. The incidence of EML4-ALK in non-small cell lung cancer patients is about 4% to 5%, and the expression rate in ade...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156C12Q2600/166
Inventor 李南南易吉朱师达罗慧娟陈小芳
Owner SHENZHEN HUADA GENE INST
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