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Oligonucleotide, method and kit for detecting ETV6-RUNX1 fusion gene in sample

A technology of ETV6-RUNX1 and ETV6-F, applied in the fields of life science and biology, can solve the problems of high cost and inferior specificity, and achieve the effect of simple operation, prediction of prognosis, and prevention of clinical recurrence.

Pending Publication Date: 2021-03-02
FUZHOU ADICON CLINICAL LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, because SYBR Green I is an unsaturated dye, its specificity is not as good as that of the double-probe hybridization method and TaqMan method, and its specificity must be judged by observing the melting curve; while the double-probe hybridization method is more expensive

Method used

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  • Oligonucleotide, method and kit for detecting ETV6-RUNX1 fusion gene in sample
  • Oligonucleotide, method and kit for detecting ETV6-RUNX1 fusion gene in sample
  • Oligonucleotide, method and kit for detecting ETV6-RUNX1 fusion gene in sample

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Experimental program
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Effect test

Embodiment 1

[0048] The invention is a method for assisting clinical typing diagnosis of B-ALL and formulation of an individualized treatment plan. It mainly includes the following reagents: erythrocyte lysate, its formula is: 16μmol / L ammonium chloride, 1mmol / L potassium bicarbonate and 12.5μmol / LEDTA; sample RNA extraction solution: TRIzol, chloroform, isopropanol, 75% ethanol and RNase -free water; detection system PCR reaction solution: ReverTra Ace qPCR RT Kit (TOYOBO); THUNDERBIRD Probe qPCR Mix (2×), primers ABL-F, ABL-R and probes ABL-Probe and ETV6- The primers ETV6-F, RUNX1-R and the probe ETV6-probe of the RUNX1 target gene were all 10 μM;

[0049] Wherein the primers and probes of internal reference gene ABL and target gene ETV6-RUNX1 are detected, as described in Table 1:

[0050] Table 1. Primer and Probe Sequences

[0051]

[0052] Positive control substance: a plasmid solution containing the ETV6-RUNX1 cDNA sequence (ie, a positive plasmid containing the ETV6-RUNX1 cDN...

Embodiment 2

[0057] The operation process of the inventive method:

[0058] (1) Extraction of total RNA in blood: Add 1ml of erythrocyte lysate into a clean 1.5ml centrifuge tube, take 0.5ml of anticoagulated blood and mix well. Let stand at room temperature for 10 minutes; centrifuge at 1500rpm for 5min, discard the supernatant, and collect the cells at the bottom; add 0.5ml red blood cell lysate again, centrifuge at 1500rpm for 5min, discard the supernatant, and collect the cells at the bottom; add 1ml TRIzol to the cells, and pipette repeatedly until the precipitation is complete Dissolve, let stand at room temperature for 5 minutes; add 0.2ml chloroform, shake evenly; centrifuge at 14000rpm at 4°C for 10 minutes, absorb the supernatant layer and transfer to another new centrifuge tube; add an equal volume of isopropanol, mix well up and down, and let stand at room temperature for 10 minutes Centrifuge at 14000rpm at 4°C for 10min, discard the supernatant, add 1ml of 75% ethanol, wash t...

Embodiment 3

[0068] Detection sensitivity with nucleic acid detection method of the present invention:

[0069] Calculate the original copy number of the ETV6-RUNX1 positive plasmid according to the copy number calculation formula, and design several dilutions on this basis to obtain a copy number of 10 10 、10 9 、10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 , 10, 1copies / μl positive plasmid, with 10 2 , 10, and 1 copy number of plasmids were used as templates for PCR amplification, and 10 replicate holes were made for each concentration. The results are shown in Table 2, figure 2 It is the amplification curve chart of ETV6-RUNX1 positive plasmid sensitivity detection. It can be seen from Table 2 that all the plasmids with 100 copies / μL can be amplified, while the plasmids with 10 copies / μL are only partially amplified. Therefore, the lower limit of detection of the ETV6-RUNX1 plasmid in the present invention is 100 copies / μL.

[0070] Table 2 Ct value of sensitivity detection of ET...

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Abstract

The invention relates to an oligonucleotide, method and kit for detecting ETV6-RUNX1 fusion gene in a sample. The kit mainly comprises an upstream primer ETV6-F, a downstream primer RUNX1-R and a probe ETV6-Probe for detecting the ETV6-RUNX1 fusion gene, and an upstream primer ABL-F, a downstream primer ABL-R and a probe ABL-Probe for detecting ABI reference gene. The kit is used for detecting thecondition of the ETV6-RUNX1 fusion gene in-vivo acute B lymphocytic leukemia patients, and can conveniently carry out related auxiliary diagnosis and treatment.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and is specially designed to provide oligonucleotides, methods and kits for detecting ETV6-RUNX1 fusion genes in samples, and uses probe real-time fluorescent PCR technology to detect ETV6-RUNX1 in human B-ALL. RUNX1 fusion gene was detected. Background technique [0002] The occurrence of leukemia is a complex and multi-step process. Chromosomal mutations can form fusion genes with tumor characteristics, and can also cause uncontrolled expression of some genes that play an important role in the regulation of cell growth and apoptosis, thereby interfering with cell proliferation and differentiation. The normal regulation pathway of , maturation and apoptosis, ultimately leading to leukemogenesis. Leukemia is the most common malignant tumor disease in childhood, with an incidence rate of 3-5 / 100,000, and it is also a disease that seriously threatens children's health. Acute lymphobl...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/6886C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/156C12Q2600/166C12Q2600/118C12Q2531/113C12Q2527/143C12Q2521/107C12Q2561/101C12Q2545/114
Inventor 刘赵玲王淑一
Owner FUZHOU ADICON CLINICAL LAB INC
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