Oligonucleotide, method and kit for detecting ZNF 198-FGFR1 fusion gene in sample

A ZNF198-FGFR1 and ZNF198-FGFR1-F technology, which is applied in the field of oligonucleotides and kits for detecting ZNF198-FGFR1 fusion genes in samples, can solve the problems of patients losing optimal treatment timing and lack of sufficient knowledge, etc. To achieve the effect of eliminating the need for conditional exploration, saving detection time, and improving experimental efficiency

Inactive Publication Date: 2017-11-24
南京艾迪康医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] It is very important to use targeted drugs in the early stage of the disease for the treatment of such diseases. At present, there are drugs targeting EMS with FGFR1 gene rearrangement, but due to the lack of sufficient understanding of this type of disease, many patients lose their The best time to treat

Method used

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  • Oligonucleotide, method and kit for detecting ZNF 198-FGFR1 fusion gene in sample
  • Oligonucleotide, method and kit for detecting ZNF 198-FGFR1 fusion gene in sample
  • Oligonucleotide, method and kit for detecting ZNF 198-FGFR1 fusion gene in sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Oligonucleotides for detecting the ZNF198-FGFR1 fusion gene in the sample, the oligonucleotides include the upstream primer ZNF198-FGFR1-F for detecting the ZNF198-FGFR1 fusion gene, the downstream primer ZNF198-FGFR1-R and the probe ZNF198- FGFR1-Probe, its base sequence is:

[0045] ZNF198-FGFR1-F:TGCCTATCCCTGTGCCTG;

[0046] ZNF198-FGFR1-R:GAGTCCCACTGGAGGAGAGC;

[0047] ZNF198-FGFR1-Probe: FAM-TGATGGCCGAACCAGAAGAACCC-TAMRA.

[0048] Further, the oligonucleotide also includes an upstream primer abl-F, a downstream primer abl-R and a probe abl-Probe for detecting the abl internal reference gene, and its base sequence is:

[0049] abl-F:GATACGAAGGGAGGGTGTACCA;

[0050] abl-R:CTCGGCCAGGGTGTTGAA;

[0051] abl-Probe: FAM-GCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA.

[0052] A method for detecting ZNF198-FGFR1 fusion gene in a sample, the steps of which are:

[0053] (1) extract the RNA in the sample;

[0054] (2) Reverse transcribe the RNA extracted in (1) into cDNA;

[005...

Embodiment 2

[0076] Embodiment 2 detection steps

[0077] According to the oligonucleotide, method and kit in Example 1, detect the ZNF198-FGFR1 fusion gene in the sample (such as blood), the steps are as follows:

[0078] (1) Extract tissue RNA from blood:

[0079] Add 1ml of erythrocyte lysate to a clean 1.5ml centrifuge tube, take 0.5ml of anticoagulated blood and mix well, let it stand at room temperature for 10min; centrifuge at 5000rpm for 5min, discard the supernatant, and collect the cells at the bottom; add 0.5ml of erythrocyte lysate again, Centrifuge at 5000rpm for 5min, discard the supernatant, and collect the cells at the bottom; add 1ml TRIzol to the cells, pipette repeatedly until the precipitate is completely dissolved, and let stand at room temperature for 5min; add 0.2ml chloroform, shake evenly; centrifuge at 14000rpm 4°C for 10min, absorb the supernatant and transfer Transfer to another new centrifuge tube; add an equal volume of isopropanol, mix well up and down, let ...

Embodiment 3

[0089] Embodiment 3: Sensitivity test result

[0090] With the positive control substance in embodiment 1, about to contain the plasmid of ZNF198-FGFR1cDNA sequence, according to 10,10 2 ……..10 10 The dilution multiples of each step were used for gradient dilution, and the dilutions obtained by each gradient dilution were respectively used as cDNA templates, and fluorescent PCR amplification and detection were carried out according to the steps (3) to (7) in Example 2. Repeat the detection 10 times in the well, dilute 10 10 and 10 11 The test results are shown in Table 1 and Table 2 respectively:

[0091] Table 1 Dilution 10 10 When (concentration is 10copies / μL) detection results

[0092]

[0093]

[0094] Table 2 Dilution 10 11 When (concentration is 1copies / μL) detection results

[0095] Wells of 96-well plate

Ct(ZNF198-FGFR1)

A9

39.32

B9

39.94

C8

Undet.

C9

Undet.

D8

Undet.

D9

Undet.

E8...

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Abstract

The invention discloses a kit for detecting a ZNF 198-FGFR1 fusion gene in a patient with 8p11 EMS. The kit comprises a primer for amplifying the ZNF 198-FGFR1 fusion gene and a probe, and a primer and a probe of a reference gene ABL. According to the kit, the existence of the ZNF 198-FGFR1 fusion gene and the expression amount of the ZNF 198-FGFR1 fusion gene in the body of the patient with the EMS can be detected quickly. The detection result completed by utilizing the kit is accurate and flexible, can diagnose the EMS in an assistant manner and determine a fusion type, has a strong guiding function on using a targeted target medicament in the early stage of disease, and has important significance in performing in-time intervention treatment, regulating a treatment scheme, evaluating a treatment effect and predicting prognosis.

Description

technical field [0001] The present invention provides oligonucleotides, methods and kits for detecting ZNF198-FGFR1 fusion gene in samples, using probe Taqman real-time fluorescent quantitative PCR technology for detecting ZNF198-FGFR1 fusion in human 8p11 myeloproliferative syndrome (EMS) patients At the same time, more accurate screening of high-risk groups is carried out. The kit can effectively save detection time and improve detection accuracy. Background technique [0002] 8p11 myeloproliferative syndrome (EMS) is a very rare aggressive hematological neoplasm with rearrangement of the FGFR1 gene, which localizes to 8p11 of myeloid and lymphoid cells. In the latest revised WHO classification of hematopoietic and lymphoid tumors in 2008, 8p11 / FGFR1 rearrangement was used as a specific classification marker, and EMS was officially named as myeloid and lymphoid tumors with FGFR1 abnormalities. The main biological features of hematological malignancies with FGFR1 rearrang...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q1/686C12Q2600/118C12Q2600/166
Inventor 林筱剑黄开新王淑一
Owner 南京艾迪康医学检验所有限公司
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