BCR gene and ABL gene detection probe, preparation method thereof and reagent kit

A gene detection and gene technology, applied in the field of BCR gene and ABL gene detection probes and their preparation, can solve the problems of lack of specificity, high detection kits, etc., achieve good discrimination, good repeatability of results, and improve survival rate Effect

Inactive Publication Date: 2016-04-13
GUANGZHOU LBP MEDICINE SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, for BCR / ABL gene FISH detection, there is still a lack of highly specific detection kits

Method used

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  • BCR gene and ABL gene detection probe, preparation method thereof and reagent kit
  • BCR gene and ABL gene detection probe, preparation method thereof and reagent kit
  • BCR gene and ABL gene detection probe, preparation method thereof and reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0038] The preparation of embodiment 1BCR gene and ABL gene detection probe

[0039]The preparation method of described ABL detection probe (GSPABL), comprises the following steps:

[0040] Clone screening: select clones containing the target gene ABL and both ends of the sequence, such as Figure 1A shown.

[0041] GSPABL1 includes a first probe, a second probe, a third probe, a fourth probe, and a fifth probe, which may be one, several, or a combination of multiples.

[0042] In this embodiment, the combination of five probes was selected, as shown in the following table, which were purchased from Invitrogen RP11BAC and CTDBAC clone libraries.

[0043] ABL1 gene chr9:133,589,268-133,763,062,173,795bp

[0044] Probe set 1

BAC

Insert segment start and end position

3 --> first probe

CTD-2037L19

chr9:133263327…133469440(206Kb)

second probe

RP11-21G10

chr9:133463380…133642562(179Kb)

third probe

CTD-2526G20

chr9:133,...

Embodiment 2

[0061] Embodiment 2: BCR gene and ABL gene detection kit preparation method

[0062] BCR gene and ABL gene detection kit include two components of BCR / ABL hybridization solution and DAPI counterstaining agent, wherein BCR and ABL hybridization solution comprise a group of GSPABL gene probes and two groups of GSPBCR gene probes described in Example 1 combination of needles. The BCR gene has two groups of detection probes, and the ABL gene has a group of detection probes. In the present embodiment, the BCR gene and the ABL gene detection kit are: BCR (group 2+group 3)+ABL (group 1) DAPI counterstaining agents such as combinations, buffer components used in hybridization environment (promoting hybridization), and COTHumanDNA with closed repeat sequences are mainly used for cell counterstaining after hybridization, in which DAPI will bind to DNA, making the cell nucleus appear blue For fluorescence, a counterstain containing p-phenylenediamine can keep the fluorescence stable.

...

Embodiment 3

[0070] Embodiment 3: the detection method of BCR gene and ABL gene detection kit

[0071] 1. Sample processing

[0072] 1.1 Take 2-3ml of peripheral blood or bone marrow to be tested (anticoagulated with sodium heparin) and centrifuge at 2000rpm for 5min, carefully remove the supernatant.

[0073] 1.2 Add 10ml of hypotonic solution (0.075mol / LKCl), mix by pipetting, and let stand for 3min.

[0074] 1.337 ± 1 ℃ water bath box hypotonic 30min.

[0075] 1.4 Add 1ml of fresh fixative, mix by pipetting, and pre-fix at room temperature for 10min.

[0076] 1.5 Mix by pipetting and centrifuge at 2000rpm for 5min.

[0077] 1.6 Remove the supernatant, add 5-10ml of fresh fixative to the sediment, mix by pipetting, and let stand at room temperature for 10min.

[0078] 1. Centrifuge at 72000rpm for 5min, remove the supernatant.

[0079] 1.8 The above washing steps can be repeated until the cell pellet is washed white (this step does not need to stand at room temperature for 10 minute...

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Abstract

The invention relates to a BCR gene and ABL gene detection probe and a preparation method thereof. The method includes the following steps that a selected BAC clone for a BCR gene is at least one of RP11-1026A5, RP11-165G5 and CTD-2079I4, a selected BAC clone is at least one of CTD-2302P22, CTD-2037J11 and CTD-2509L4, a selected BAC clone for an ABL is at least one of CTD-2037L19, RP11-21G10 and CTD-2526G20, the DNA of a plasmid is obtained, and marking is conducted. The invention further discloses a reagent kit provided with the BCR gene and ABL gene detection probe. By obtaining the optimal BCR gene and ABL detection probe through screening, signal line counting is accurate and quick, and result repeatability is good.

Description

technical field [0001] The invention belongs to biotechnology, and in particular relates to a BCR gene and ABL gene detection probe, a preparation method and a test kit thereof. Background technique [0002] Chronic myelogenous leukemia (chronic myelogenous leukemia, CML) is a clonal myeloproliferative neoplasm originating from hematopoietic stem cells, mainly involving the granulocyte lineage, and manifested as a persistent and progressive increase in the number of peripheral blood leukocytes. The onset of CML is slow, and the initial symptoms are not obvious. The natural course of the disease progresses from the chronic phase to the accelerated phase, and finally to the blast phase. After the blast, patients often die within 3 to 5 months. [0003] More than 90% of leukemia cells in patients have constant and characteristic Ph chromosome and its molecular marker BCR / ABL fusion gene. Most of the Ph chromosomes are t(9;22)(q34;q11) typical translocations, and a few patient...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12N15/11C12Q1/68C12Q1/6886C12Q2600/106C12Q2600/166
Inventor 何瑰陈绍宇张会清
Owner GUANGZHOU LBP MEDICINE SCI & TECH
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