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A method for detecting eml4-alk, ros1 and ret fusion gene mutations

A gene and unfused technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of poor detection specificity, large difference in result interpretation, low sensitivity, etc., to achieve high specificity and avoid pollution effect

Active Publication Date: 2021-09-17
XUKANG MEDICAL SCI & TECH (SUZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection sensitivity of immunohistochemistry (IHC) is poor, only about 10%; the detection specificity of FISH method is poor, the sensitivity is low, the result interpretation is different, the detection time is long, and it is impossible to detect multiple fusion variants produced by RET fusion gene at the same time And expensive special equipment is needed, the cost of reagents is high, and the operation is complicated; conventional RT-PCR method is sensitive and objective compared with FISH and IHC, but it still cannot meet the actual needs of clinical detection, and its sensitivity and specificity need to be further improved. The detection will lead to the occurrence of missed detection and false negative

Method used

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  • A method for detecting eml4-alk, ros1 and ret fusion gene mutations
  • A method for detecting eml4-alk, ros1 and ret fusion gene mutations
  • A method for detecting eml4-alk, ros1 and ret fusion gene mutations

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Detection sample processing and RNA extraction

[0088] 1. Test samples include fresh pathological tissue, frozen pathological sections, paraffin-embedded tissue or sections.

[0089] a. In the case of using cells as input, for fresh pathological tissue, first perform cell dispersion treatment, that is, grind the tissue in liquid nitrogen, add about 600 μL of PBS per 20-30 mg of tissue, and centrifuge at 12,000 rpm (~13,400 × g) After 1 minute, the cell pellet was obtained, the supernatant was discarded, the cell pellet was aspirated, and the cell suspension was diluted with 1x PBS to a density of ~500 cells / μL

[0090] b. In the case of using total RNA as input, take about 1 gram from fresh pathological tissue, frozen pathological sections, paraffin-embedded tissue or sections, and use Qiagen's RNA extraction kit to extract the RNA. Tissues were paraffin-embedded and their RNA was extracted using Qiagen's FFPE tissue RNA extraction kit. The above-mentioned ...

Embodiment 2

[0096] Example 2 Reverse transcription to obtain double-stranded full-length cDNA

[0097] 1. Pipette 1.7*N μL of RT Enzyme Mix into the RT Buffer of the previous step, mix by hand, and centrifuge;

[0098] 2. Add 15μL of RT Buffer and RT Enzyme Mix to each cell lysate, centrifuge briefly and place on ice immediately, and incubate the samples on a preheated PCR machine under the following conditions:

[0099]

[0100] Example 3 Amplification

[0101] Add 29.25 μL PCR Mix to the reverse transcription product (the solution volume is 49.25 μL at this time), add 0.75 μL Index primer to each reaction tube, mix well and centrifuge, and amplify in the PCR machine. The reaction conditions are as follows:

[0102]

Embodiment 4

[0103] Example 4 Library Detection

[0104] Step 1: Purification

[0105] 1) Transfer the amplification product to a centrifuge tube, take 0.8× (40uL) Ampure XP magnetic beads or CMpure magnetic beads, mix with the amplification product and place it on a magnetic stand for 10 minutes;

[0106] 2) After all the magnetic beads are adsorbed on the tube wall (about 5 min), discard the supernatant and wash the magnetic beads twice with newly prepared 80% ethanol, and discard the supernatant;

[0107] 3) Stand at room temperature for 5 minutes, and after the magnetic beads are dry (please be careful not to over-dry the magnetic beads to cause the magnetic beads to crack, so as not to affect the recovery efficiency), resuspend the magnetic beads with 17.5uL TE buffer, EB buffer or nucleic acid-free water according to downstream needs;

[0108] 4) After standing at room temperature for 5 minutes, place the centrifuge tube on a magnetic stand, and aspirate 15uL of the supernatant, whi...

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Abstract

The invention relates to the field of biotechnology, in particular to a method for one-time detection of fusion gene mutations of EML4‑ALK, ROS1 and RET through library construction and sequencing. The present invention can start from 1-2000 fresh tissue cells, 10pg-20ng extracted total RNA, or plasma free RNA within 3 hours, under the action of ALK, ROS1, RET specific primers and reverse transcriptase Reverse transcribe the mRNA containing the fusion type information and obtain the first strand of cDNA, then add a linker sequence (with a molecular tag) to the 3' end of the cDNA by template switching technology and generate the second strand of cDNA, and then use the linker region The segment is the primer anchor site for exponential amplification and Illumina library adapters are added to both ends of the cDNA to obtain a high-quality cDNA library that meets the requirements of downstream analysis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for one-time detection of EML4-ALK, ROS1 and RET fusion gene mutations, the method comprising constructing a library and sequencing. Background technique [0002] Lung cancer is the most common malignant tumor worldwide, and its morbidity and mortality ranks first among all cancers. Every year, more than 1.3 million people die from lung cancer worldwide, nearly half of them in developing countries. According to the statistics of the Ministry of Health of my country in 2010, the mortality rate of lung cancer was 30.83 / 100,000, and lung cancer has become the first malignant tumor in morbidity and mortality (Orras JM, Fernandez E, Gonzalez JR, et al. Lung cancer mortality in European regions (1955-1997). Ann Oncol, 2003, 14(1): 159_161; Ministry of Health of the People's Republic of China, "2010 China Health Statistics Yearbook). [0003] Lung cancer can be divided into two ty...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C12N15/11
CPCC12Q1/6869C12Q2521/107C12Q2535/122C12Q2525/191
Inventor 胡春旭陆思嘉任军
Owner XUKANG MEDICAL SCI & TECH (SUZHOU) CO LTD
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