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Gene probe composition for detecting acute promyelocytic leukemia, kit and application

A promyelocytic and gene probe technology is applied in the field of gene probe compositions for detecting acute promyelocytic leukemia, and achieves the effect of simplifying operation and overcoming market gaps

Inactive Publication Date: 2018-06-29
UNION STEMCELL & GENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fluorescence in situ hybridization has the advantages of safety, rapidity, high sensitivity, strong detection signal, high hybridization specificity, and the ability to display multiple colors at the same time, but the FISH diagnostic kit currently used in the molecular diagnosis of acute promyelocytic leukemia (APL) Still blank in the domestic market

Method used

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  • Gene probe composition for detecting acute promyelocytic leukemia, kit and application
  • Gene probe composition for detecting acute promyelocytic leukemia, kit and application
  • Gene probe composition for detecting acute promyelocytic leukemia, kit and application

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preparation example Construction

[0054] 3), preparation and verification of the gene probe: after obtaining the clone of the target gene, a large amount of DNA was extracted, and the concentration and purity (260nm / 280nm) of the plasmid were measured with an ND-1000 ultra-micro spectrophotometer; Plasmid DNA is fluorescently labeled, (choose the best fluorescent dye and the filter matching combination of the fluorescent microscope for microscopic examination, so that the detected fluorescent signal intensity is the largest); the labeled product is precipitated and concentrated; the fluorescently labeled probe Dissolve in hybridization buffer containing 50% deionized formamide, store at -20°C in the dark, and use normal human peripheral blood mononuclear cells for probe identification.

[0055] In a superior embodiment of the present invention, the clones containing the optimal PML gene in the clone screening step are numbered RP11-8P11, RP11-243B22, RP11-161C1 (UCSC genome browser);

[0056] In an advantageou...

Embodiment 1

[0091] Embodiment 1: The preparation method of PML / RARα gene probe composition comprises the following steps

[0092] 1. Clone screening:

[0093] By searching UCSC genome browser, NCBI Clone Registry, Ensembl GenomeBrowser and other databases, all clones containing PML / RARα genes were searched. The optimal clones containing this gene were screened out, and the PML numbers were: RP11-8P11, RP11-243B22, RP11-161C1; the RARα numbers were RP11-58E20, RP11-48010, RP11-465J4. (As shown in Table 1)

[0094] Table 1 PML, RaRα gene detection kit gene fragment markers

[0095]

[0096] Cultivation and identification of clones: Purchase clones (Invitrogen, USA) according to the clone number, add 8 microliters of clone bacteria liquid to 5 milliliters of LB liquid medium containing chloramphenicol resistance, shake and activate at 37°C for 12 hours; then All the bacterial liquid was added to 450 ml of LB liquid medium containing chloramphenicol resistance, and the bacterial liquid ...

Embodiment 2

[0146] Embodiment 2: a kind of acute promyelocytic leukemia fluorescence in situ hybridization detection kit (50 parts), the composition is as follows:

[0147] 1) Fluorescence-labeled probe set hybridization mixture 100 microliters; 1 tube

[0148] 2) DAPI counterstain solution 500 microliters; 1 tube

[0149] 3) 20ml of 20XSSPE washing solution; 1 bottle

[0150] 4) Instruction manual 1 copy

[0151] Wherein the concentration of the PML gene probe in the hybridization mixture of the fluorescently labeled probe group is: 4ng / μl, and the concentration of the RARα gene probe is: 4ng / μl.

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Abstract

The invention discloses a gene probe composition for detecting acute promyelocytic leukemia, a kit and application. The gene probe composition comprises a PML gene probe and a RaR alpha gene probe, wherein the PML gene probe includes RP11-8P11, RP11-243B22 and RP11-161C1; the RaR alpha gene probe includes RP11-58E20, RP11-48O10 and RP11-465J4. The PML gene probe and the RaR alpha gene probe are fluorescein marked probes. The kit can be used for performing molecular pathological classification on the acute promyelocytic leukemia. The gene probe composition can be directly used for fluorescencehybrid method detection of medium-period or interphase acute promyelocytic leukemic cells; the cell culture and the preparation of high-quality medium-period chromosome flakes are not needed; the operation is greatly simplified.

Description

technical field [0001] The invention relates to the field of biological genetics, in particular to a gene probe composition, kit and application for detecting acute promyelocytic leukemia. Background technique [0002] Acute promyelocytic leukemia (APL) is a hematological malignancy and a special type of acute myeloid leukemia (AML), which is designated as acute myeloid leukemia type M3 by the FAB collaboration group. It is characterized by the unlimited proliferation of a large number of leukemia cells in the bone marrow and other hematopoietic tissues, and enters the peripheral blood, while the production of normal blood cells is significantly inhibited. However, more than 95% of acute promyelocytic leukemia has PML / RARα fusion gene. Molecular biology studies have shown that the fusion gene is a specific molecular marker of APL with a high degree of specificity and is an important mechanism for the pathogenesis of APL patients. [0003] At present, the diagnosis of vario...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6841C12N15/11
CPCC12Q1/6886C12Q1/6841C12Q2600/118C12Q2600/158C12Q2563/107
Inventor 缪为民李思奇李娟
Owner UNION STEMCELL & GENE ENG
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