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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 melting curves, which is applied in the determination/inspection of microorganisms, recombinant DNA technology, biochemical equipment and methods, etc., can solve the difficulties of multiple fusion gene types, many changes in fusion gene sequences, and mixed interstitial cell components and other problems, to achieve the effect of short detection cycle, low detection cost and fast speed

Active Publication Date: 2022-06-07
THE SECOND HOSPITAL OF DALIAN MEDICAL UNIV
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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] Twenty kinds of ALK fusion gene standard product melting curve, second derivative curve and key parameter value acquisition, the specific steps are as follows:

[0070] 1. Preparation of positive samples for different types of ALK fusion genes

[0071] 1.1 Preparation of amplification template

[0072] (1) RNA extraction: The total RNA of lung cancer cell lines A549, H1975, H2228, and H3122 was extracted and isolated by using the peripheral blood and cultured cell RNA extraction kits (Roche's high-purity RNA extraction kit), among which A549, H1975 ALK fusion gene negative cell lines, H2228, H3122 fusion gene positive cell lines, 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 260 nm, 280 nm and 230 nm to judge the concentration and purity of RNA samples; for qualified RNA samples, use the Biobio cDNA First Strand Synthesis Kit to synthesize cDNA by reverse transcription , ...

Embodiment 2

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

[0115] 1. Clinical specimens:

[0116] 52 cases of lung adenocarcinoma paraffin tissue were collected by double-blind method. Ventana immunohistochemical detection system was used to detect ALK fusion gene (ALK monoclonal antibody clone number: D5F3, Ventana company). One case of pleural effusion specimens was obtained from patients with advanced lung adenocarcinoma who had not undergone surgical treatment, radiation or chemotherapy.

[0117] 2. RNA extraction

[0118] 2.1 Extraction of total RNA 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 RNase-free, DNase-free, steri...

Embodiment 3

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

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

[0145] 2. Quantify and dilute the cDNA samples of A549, H2228 and H3122 by using the internal reference gene (GAPDH) standard curve method, so that the three internal reference genes have the same expression. The cDNA samples of H2228 and H3122 were diluted with the A549 cDNA samples with the same internal reference gene expression, and the final mass percentage concentrations of the cDNAs of H2228 and H3122 in the mixed samples were 100%, 90%, 80%, 70%, 60%, and 50%. , 40%, 30%, 20%, 10%, 5%, 2.5%, 1%, 0%. ...

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Abstract

The invention discloses an ALK fusion gene detection and typing kit based on high-resolution melting curve analysis of a sandwich method. By adding a low GC content linker at the 5' end of the gene-specific primer, the target fragment of the sandwich sequence is amplified segmentally, including fusion gene fragments with high and low GC content consensus sequences and variable sequences. The general analysis method of high-resolution melting curve can determine the presence or absence of fusion genes, and the application of fluorescence intensity-temperature second-order derivative curve combined with key parameter analysis methods can realize digital judgment of fusion gene types. The invention solves the problems of high cost, long cycle and difficult typing of ALK fusion gene detection in lung cancer at present, and integrates the advantages of real-time quantitative reverse transcription PCR and high-resolution melting curve analysis, and can be applied to fresh tissues, paraffin wax Embedded tissue, pleural effusion and other types of specimens are fused with genetic testing.

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 ALK fusion genes of common types of 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 my 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 these drug-sensitive patients have been significantly improved. At present, the target of molecular targeted drug tyrosine kinase inhibitor (TKI) for lung cancer mainly targets two types of gene mutations, one is point mutation and deletion mutation of EGFR and other genes, and the other is fusion gene. Gene point mutations and deletion mutations are relatively easy to detect, but fusion genes in solid tumors such as lung c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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