Clonal eosinophilia fusion gene detection probe composition, kit and application thereof

An eosinophil detection kit technology, applied in recombinant DNA technology, microbial assay/inspection, biochemical equipment and methods, etc., can solve the problems of adding new fusions, poor scalability, and missed detection of new fusions, etc. Achieve the effect of low sequencing cost, reliable results and high sensitivity

Active Publication Date: 2021-08-13
QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is economical and simple, with high sensitivity, but for different fusions, primers need to be adjusted separately to work in the same system, and new fusions cannot be added flexibly, and the scalability is poor
In addition, this

Method used

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  • Clonal eosinophilia fusion gene detection probe composition, kit and application thereof
  • Clonal eosinophilia fusion gene detection probe composition, kit and application thereof
  • Clonal eosinophilia fusion gene detection probe composition, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] In this embodiment, fusion detection is performed on the bone marrow samples of patients with clonal hypereosinophilia.

[0063] (1) Nucleic acid extraction

[0064] Add the sample and EL buffer at a ratio of 1:5, and incubate on ice, briefly vortex and mix twice during the incubation period to lyse the red blood cells, and then incubate at 4°C at 400 ×g Centrifuge for 10 minutes, discard the supernatant; add EL buffer (2 times the volume of the sample) at a ratio of 1:2, vortex briefly to mix, 400 at 4°C ×g Centrifuge for 10 min, discard the supernatant;

[0065] Add appropriate RLT buffer solution according to the sample volume to dissolve leukocytes, vortex or pipette to mix, transfer to the lysis column, centrifuge at the maximum speed for 2 minutes, and collect the filtrate; add 1 volume of 70% ethanol to the filtrate and pipette to mix. Transfer to filter column, 8000 ×g Centrifuge for 15s, discard the collection tube; transfer the filter column to a new collec...

Embodiment 2

[0093] Example 2 Capture of related fragments of the target fusion gene

[0094] (1) Capture target fragments

[0095] Put the above total amount of 2 μg pre-library into a new 1.5 mL centrifuge tube, add 5 μL HumanCot-1 DNA and 2 μL Universal Blockers to it, cover the tube cap, and pierce a hole on the tube cap with a disposable syringe Put it into a vacuum concentrator, and dry at a constant temperature of 60°C until there is no liquid;

[0096] Add a total volume of 17 μL hybridization solution (including 2×HybridizationBuffer, Hybridization Buffer Enhancer, probe composition and Nuclease-Free Water to the dry centrifuge tube, where the concentration of the probe composition in the reaction system is 400 amol / probe / rxn), mix well, place at room temperature for 5 min, transfer to a PCR tube, and run the HYB program (hot lid at 100°C, 95°C for 30 s, 65°C for 4 h, 65°C Hold);

[0097] After the HYB program runs, transfer the washed 17 μL M-270 magnetic bead solution to the ...

Embodiment 3

[0110] In this embodiment, bone marrow samples from 6 patients with clinically suspected hypereosinophilia were used for detection.

[0111] The detection method comprises steps such as image 3 Shown: first extract the RNA in the sample, then carry out mRNA enrichment, and then carry out pre-library construction, including: RNA thermal interruption, reverse transcription of fragmented RNA into cDNA, end repair, adding A tail, adding Connecting adapters and PCR amplification; then, using the probe composition of the present invention for hybridization capture, including: blocking library DNA, washing magnetic beads, hybridization, and obtaining a capture library after elution, followed by amplification and purification to obtain On-board inspection;

[0112] The specific implementation steps are shown in Example 1 and Example 2.

[0113] The test results are shown in Table 6. Among the 6 patients with clinically suspected hypereosinophilia, MYO18A-PDGFRB fusion was detected ...

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Abstract

The invention provides a clonal eosinophilia fusion gene detection probe composition, a kit and application thereof. According to the invention, firstly, related fusion genes are classified, wherein core genes FGFR1, PDGFRA and PDGFRB for clonal eosinophilia fusion detection are I-type genes, so that full-length transcripts of the genes are used as target regions; and other genes are II-type genes, so that only fragments near the fusion breakpoint are used as target regions. According to a base complementary pairing principle, complementary sequences of target regions are designed as probes to form a 'liquid phase chip', the probes relate to 39 target genes, eosinophilia fusion genes are detected by capturing corresponding target sequences in a transcriptome library and combining high-throughput sequencing, multiple fusion can be detected at a time, new fusion can be discovered, the detection cost is reduced, and the accuracy of a detection result is improved.

Description

technical field [0001] The invention belongs to the technical field of liquid phase capture and the technical field of transcriptome sequencing analysis, and relates to a clonal hypereosinophilia fusion gene detection probe composition, a kit and an application thereof. Background technique [0002] Eosinophilia (Eosinophilia) refers to a group of diseases with increased eosinophils or tissue damage in peripheral blood, usually with an absolute count higher than 0.5×10 9 / L is standard. If the number of eosinophils in the peripheral blood is higher than 1.5×10 9 / L, regardless of whether there is organ damage, is called Hypereosinophilia (HE). Among them, eosinophils ranged from 1.5 to 5×10 9 / L, called moderate hypertrophy, >5×10 9 / L is called severe increase. Hypereosinophilic syndrome can be divided into four categories according to etiology: reactive hypereosinophilia, secondary hypereosinophilia, idiopathic hypereosinophilia, and clonal eosinophilia polycythem...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6806C12Q1/686C12Q1/6869C12N15/10
CPCC12N15/1013C12Q1/6806C12Q1/686C12Q1/6869C12Q1/6883C12Q2535/122
Inventor 顾凯丽陆亚红邓啸张亚飞
Owner QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD
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