Kit for detecting FLT3-ITD (Fms-like tyrosine kinase 3-internal tandem duplication) gene mutation by using fluorescence PCR (Polymerase Chain Reaction) capillary electrophoresis

A FLT3-ITD, kit technology, applied in the direction of microbial determination/examination, biochemical equipment and methods, etc., can solve problems such as poor prognosis, achieve improved reliability and accuracy, high sensitivity, avoid false negatives and The effect of false positives

Inactive Publication Date: 2013-05-15
DAAN GENE CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, the prognosis of different ITD patients is also different, which is related to the ITD-AR (internal tandem allelic ratio, internal tandem repeat allele ratio) value, that is, the ITD:WT (FLT3 wild-type gene) ratio, the ITD-AR value increases May lead to poor prognosis, so ITD-AR value is also a strong independent prognostic factor in AML patients

Method used

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  • Kit for detecting FLT3-ITD (Fms-like tyrosine kinase 3-internal tandem duplication) gene mutation by using fluorescence PCR (Polymerase Chain Reaction) capillary electrophoresis
  • Kit for detecting FLT3-ITD (Fms-like tyrosine kinase 3-internal tandem duplication) gene mutation by using fluorescence PCR (Polymerase Chain Reaction) capillary electrophoresis
  • Kit for detecting FLT3-ITD (Fms-like tyrosine kinase 3-internal tandem duplication) gene mutation by using fluorescence PCR (Polymerase Chain Reaction) capillary electrophoresis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: detection kit and its use

[0035] 1. Prepare a kit comprising the following components: ITD PCR reaction solution A (containing SEQ ID NO: 1 and SEQ ID NO:: 2) (500 μl / tube), ITD PCR reaction solution B (75 μl / tube), ITD Positive quality control (50μl / tube), negative quality control (50μl / tube).

[0036] 2. Specimen collection, transportation and storage:

[0037] (1) Specimen collection: The specimen is peripheral blood or bone marrow. Take 3-5ml of venous blood or bone marrow from the subject, inject it into a glass tube containing EDTA (disodium ethylenediaminetetraacetic acid) or sodium citrate anticoagulant, and immediately invert the glass tube gently for 5-10 times to mix the anticoagulant. The coagulant is thoroughly mixed with the venous blood.

[0038] (2) Storage: It can be detected immediately, stored at 4°C for one week, and stored at -20°C for up to one year.

[0039] (3) Transportation: Specimens should be transported using 0°C curling....

Embodiment 2

[0059] Example 2: Detection of FLT3-ITD in AML patients by QF-PCR amplification of the FLT3 locus

[0060] Blood or bone marrow samples from AML patients were subjected to DNA extraction and purification according to the standard procedure of Qiagen DNA extraction kit. Use the ITD PCR reaction solution A and ITD PCR reaction solution B provided in the kit to carry out the amplification reaction and load the sample for analysis according to the kit detection protocol. The analysis spectrum of the sample loading results is as follows: image 3 , Figure 4 . Wild-type and ITD loci on the FLT3 gene as shown. The ITD mutation in the FLT3 gene will produce an amplified band larger than the wild-type fragment. Thus, FLT3-ITD can be rapidly detected.

Embodiment 3

[0061] Example 3: Detection of FLT3-ITD in MDS patients by QF-PCR amplification of the FLT3 locus

[0062] Blood or bone marrow samples from MDS patients were subjected to DNA extraction and purification according to the standard procedure of Qiagen DNA extraction kit. Use the ITD PCR reaction solution A and ITD PCR reaction solution B provided in the kit to carry out the amplification reaction and load the sample for analysis according to the kit detection protocol. The analysis spectrum of the sample loading results is as follows: Figure 5 . Wild-type and ITD loci on the FLT3 gene as shown. The ITD mutation in the FLT3 gene will produce an amplified band larger than the wild-type fragment. Thus, FLT3-ITD can be rapidly detected.

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Abstract

The invention relates to a kit for detecting ITD (internal tandem duplication) mutation in an FLT3 (Fms-like tyrosine kinase 3) gene juxtamembrane domain and in particular relates to a kit for detecting the FLT3-ITD in a clinical sample by using a fluorescence PCR (Polymerase Chain Reaction) capillary electrophoresis technology. The kit mainly comprises a PCR amplified reaction solution, a negative control product, a positive control product, a plurality of sealed reagent bottles or pipes which cover the kit as well as packaging boxes which are used for separating and concentratively packaging the reagent bottles or pipes, and the size and gene dosage of each amplified fragment are determined by using an electrophoresis method. The kit disclosed by the invention has high sensitivity and specificity in terms of detection and analysis of the FLT3-ITD in a whole blood or marrow sample and can be widely applied to the fields of judgment prognosis and guide treatment of leukemia and resurgence prevention of diseases.

Description

technical field [0001] The invention relates to a kit for detecting the mutation of internal tandem duplications (ITD) in the near-membrane region of FLT3 (Fms-like tyrosine kinase 3) gene, in particular to detection of FLT3-ITD in clinical samples by fluorescent PCR capillary electrophoresis technology kit. Since the kit has high sensitivity and specificity for the detection and analysis of FLT3-ITD in whole blood or bone marrow samples, it can be widely used in many fields such as judging the prognosis of leukemia, guiding treatment and predicting disease recurrence. Background technique [0002] FLT3 (Fms-like tyrosine kinase 3) is a member of the type III receptor tyrosine kinase family, and its mutation is closely related to the occurrence of leukemia. FLT3 and its ligand (FL) play an important regulatory role in the proliferation and differentiation of hematopoietic stem / progenitor cells. The binding of FL to the extracellular domain of FLT3 mediates a series of intr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 李明陈嘉昌陈华云高云程钢何蕴韶
Owner DAAN GENE CO LTD
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