PML gene and RARA gene detection probe, preparation method thereof and reagent kit

A gene detection and gene technology, which is applied in the field of PML gene and RARA gene detection probes and their preparation, can solve the problems of high detection kits and lack of specificity, so as to improve the survival rate, good discrimination, and improve the overall survival period Effect

Inactive Publication Date: 2016-03-23
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, there is still a lack of specific detection kits for PML / RARA gene FISH detection

Method used

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  • PML gene and RARA gene detection probe, preparation method thereof and reagent kit
  • PML gene and RARA gene detection probe, preparation method thereof and reagent kit
  • PML gene and RARA gene detection probe, preparation method thereof and reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The preparation of embodiment 1PML gene and RARA gene detection probe

[0037]The preparation method of the PML / RARA detection probe described in this implementation includes the following steps: selecting clones containing the target genes PML and RARA and the sequences at both ends, as shown in FIGS. 1 and 2 . GSPPML includes the first probe, the second probe, the third probe and the fourth probe, as shown in the table below, which were purchased from Invitrogen RP11BAC and CTDBAC clone libraries. GSPRARA includes the first probe, the second probe, the third probe, the fourth probe and the fifth probe, as shown in the table below, which were purchased from Invitrogen RP11BAC and CTDBAC clone libraries.

[0038] The following two groups of detection probes were prepared respectively.

[0039] PML

[0040]

BAC

Insert segment start and end position

first probe

RP11-832J18

chr15:73959323..74146088(187Kb)

second probe

CTD-252...

Embodiment 2

[0053] Embodiment 2: PML and RARA gene detection kit preparation method

[0054] PML and RARA gene detection kit include two components of PML and RARA hybridization liquid and DAPI counterstaining agent, wherein PML and RARA hybridization liquid comprise GSPPML described in embodiment 1 and GSPRARA gene probe, are used for hybridization environment (promoting Hybridization) buffer components, COT Human DNA with closed repeat sequences, etc. DAPI counterstaining agent is mainly used for cell counterstaining after hybridization, in which DAPI will bind to DNA, making the nucleus show blue fluorescence, and the counterstaining agent containing p-phenylenediamine can keep the fluorescence stable.

[0055] The specific formula is as follows:

[0056] Hybridization solution preparation

[0057]

[0058]

[0059] a. DAPI counterstain preparation

[0060] Dissolve 10 mg of p-phenylenediamine in 1 ml of PBS, adjust the pH to 9.0, add 9 ml of glycerin, shake and mix repeatedly...

Embodiment 3

[0063] Embodiment 3: the detection method of PML / RARA gene detection kit

[0064] 1. Sample processing

[0065] 1.1 Take 2-3ml of peripheral blood or bone marrow (anticoagulated with sodium heparin) and centrifuge at 2000rpm for 5min, carefully remove the supernatant.

[0066] 1.2 Add 10ml of hypotonic solution (0.075mol / LKCl), mix by pipetting, and let stand for 3min.

[0067] 1.337 ± 1 ℃ water bath box hypotonic 30min.

[0068] 1.4 Add 1ml of fresh fixative, mix by pipetting, and pre-fix at room temperature for 10min.

[0069] 1.5 Mix by pipetting and centrifuge at 2000rpm for 5min.

[0070] 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.

[0071] 1. Centrifuge at 72000rpm for 5min, remove the supernatant.

[0072] 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 minutes).

[0073] 2. Prod...

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Abstract

The invention relates a PML gene and RARA gene detection probe and a preparation method thereof. The method comprises the following steps that at least one of RP11-832J18, CTD-2529B11, RP11-756N20 and RP11-1031J4 and a selected BAC cline for an RARA gene is at least one of CTD-2360L10, RP11-737D6, CTD-3087O22, RP11-48O10 and CTD-2134K5 are included; a plasmid DNA is obtained; labeling is performed. The invention further discloses a reagent kit containing the PML gene and RARA gene detection probe for detecting acute promyelocytic leukemia PML and RARA fusion genes. The optimal PML gene and RARA gene detection probe is obtained through screening, the signal counting row is accurate and rapid, and the result repeatability is good.

Description

technical field [0001] The invention belongs to biotechnology, and in particular relates to a PML gene and RARA gene detection probe, a preparation method and a test kit thereof. Background technique [0002] Acute promyelocytic leukemia (APL) is a type of non-lymphocytic leukemia with dangerous clinical manifestations. Hemorrhage and embolism are easy to be found during onset and treatment and cause death, but it responds well to induction differentiation therapy. More than 98% of APL have a specific gene phenotype, manifested as t(15;17)(q22;q21) reciprocal translocation, and the PML / RARα fusion gene expression protein formed by the translocation has a dominant negative inhibitory effect on promyelocyte differentiation Maturation, cells are blocked at the promyelome stage, inhibiting their differentiation. In the past two decades, the clinical application of all-trans retinoic acid (ATRA) and arsenic has made APL one of the curable leukemias. [0003] Detection of PML / RA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6841C12Q1/6886C12Q2600/106C12Q2600/118C12Q2563/107C12Q2563/173C12Q2537/163C12N15/11C12Q1/68
Inventor 陈绍宇何瑰张会清
Owner GUANGZHOU LBP MEDICINE SCI & TECH
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