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ALK fusion gene detection and typing kit based on sandwich method high-resolution melting curve analysis

A technology of fusion genes and kits, which is applied in the determination/inspection of microorganisms, recombinant DNA technology, biochemical equipment and methods, etc., and can solve the difficulties of various fusion gene types, mixed interstitial cell components, and frequent changes in fusion gene sequences to achieve the effects of low pollution risk, fast detection speed, and short detection cycle

Active Publication Date: 2019-04-16
THE SECOND HOSPITAL OF DALIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many changes in the fusion gene sequence, RNA degradation is often found in paraffin-embedded tissues, interstitial cell components are mixed, etc., and it is extremely difficult to amplify multiple fusion gene types at the same time
In addition, the genotyping of high-resolution melting curve technology is mainly used to distinguish homozygous, heterozygous and other gene types. At present, the distinction of long fragments with different nucleic acid sequences mainly depends on sequencing. The application of HRMA for fusion gene detection and typing is not available at home and abroad. see the report

Method used

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  • ALK fusion gene detection and typing kit based on sandwich method high-resolution melting curve analysis
  • ALK fusion gene detection and typing kit based on sandwich method high-resolution melting curve analysis
  • ALK fusion gene detection and typing kit based on sandwich method high-resolution melting curve analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] The melting curves, second-order derivative curves, and key parameter values ​​of twenty kinds of ALK fusion gene standard products are obtained. The specific steps are as follows:

[0070] 1. Different types of ALK fusion gene positive sample preparation

[0071] 1.1 Preparation of amplification template

[0072] (1) RNA extraction: Use peripheral blood and cultured cell RNA extraction kit (Roche High Purity RNA Extraction Kit) to extract and separate the total RNA of lung cancer cell lines A549, H1975, H2228, and H3122, of which A549, H1975 ALK fusion gene-negative cell lines, H2228 and H3122 are fusion gene-positive cell lines, and the fusion types are: EML4(6)-ALK(20) and EML4(13)-ALK(20);

[0073] (2) cDNA synthesis: Use a spectrophotometer to read the absorbance values ​​at 260nm, 280nm and 230nm to judge the concentration and purity of the RNA sample; for qualified RNA samples, use Baobio cDNA first-strand synthesis kit to synthesize cDNA by reverse transcriptio...

Embodiment 2

[0114] The detection of ALK fusion gene in paraffin-embedded lung cancer tissue of clinical non-small cell lung cancer patients and pleural effusion samples of non-small cell lung cancer patients, the specific detection steps are as follows:

[0115] 1. Clinical specimens:

[0116] 52 cases of lung adenocarcinoma tissue were collected in a double-blind method, all of which had been detected by the Ventana immunohistochemical detection system to detect the ALK fusion gene (clone number of ALK monoclonal antibody: D5F3, Ventana Company). One case of pleural effusion sample was obtained from a patient with advanced lung adenocarcinoma who had not undergone surgery, radiation or chemotherapy.

[0117] 2. RNA extraction

[0118] 2.1 Total RNA extraction from paraffin tissue:

[0119] (1) For the wax block tissue detected by ALK immunohistochemistry, 2-3 slices of 10 μm thick wax rolls were continuously cut (the first 2-3 slices were discarded) and placed in a sterile 1.5mL EP tub...

Embodiment 3

[0143] Determination of the detection efficiency, detection limit and stability of fusion genotyping parameters of the ALK fusion gene detection method based on high-resolution melting curve analysis:

[0144] 1. The cDNA prepared in Example 1 was used, including the cDNA storage solution (1 mg / μL) reverse-transcribed from the total RNA of the ALK fusion gene negative lung cancer cell line A549 and the fusion gene positive cell lines H2228 and H3122, and carried out 5 consecutive times. serial dilutions.

[0145] 2. Use the internal reference gene (GAPDH) standard curve method to quantify and dilute the cDNA samples of A549, H2228 and H3122, so that the expression levels of the three internal reference genes are the same. Dilute the cDNA samples of H2228 and H3122 with the A549 cDNA sample with the same expression level of the internal reference gene, and the final mass percentage concentrations of the cDNA of H2228 and H3122 in the mixed sample are 100%, 90%, 80%, 70%, 60%, a...

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Abstract

The invention discloses an ALK fusion gene detection and typing kit based on sandwich method high-resolution melting curve analysis. A sandwich sequence target segment is amplified in a segmented manner by adding a low GC content joint at the 5' end of a gene-specific primer, and is a fusion gene fragment comprising a high and low GC content consensus sequence and a variable sequence. A high-resolution melting curve general analysis method can judge presence or absence of the fusion gene, and the fluorescence intensity-temperature second-order derivative curve is combined with a key parameteranalysis method to achieve digital judgment of the fusion gene type. The kit solves the problems of high cost, long cycle and difficult typing of the existing lung cancer ALK fusion gene detection, integrates the advantages of two technologies of real-time quantitative reverse transcription PCR and high-resolution melting curve analysis, and can be applied to fusion gene detection of fresh tissues, paraffin-embedded tissues, pleural effusion and other types of specimens.

Description

technical field [0001] The invention belongs to the technical field of in vitro detection, and in particular relates to a kit for amplifying, detecting and typing common types of ALK fusion genes in lung cancer. Background technique [0002] Lung cancer is a malignant tumor with high morbidity and mortality, and its incidence is increasing year by year in our country. The emergence of individualized molecular targeted therapy has made up for the shortcomings of surgical treatment and traditional chemotherapy, and the quality of life and survival period of such drug-sensitive patients have been significantly improved. At present, lung cancer molecular targeted drug tyrosine kinase inhibitor (TKI) mainly targets two types of gene mutations, one is point mutations and deletion mutations of EGFR and other genes, and the other is fusion genes. Gene point mutations and deletion mutations are relatively easy to detect, but fusion genes in solid tumors such as lung cancer are diffi...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/156C12Q2531/113C12Q2527/107
Inventor 李梅吕申
Owner THE SECOND HOSPITAL OF DALIAN MEDICAL UNIV
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