Method for detecting MLL-AF4 fusion gene

A technology of MLL-AF4 and fusion gene, applied in the field of life detection, can solve the problems of easy recurrence, high cost, poor repeatability, etc., and achieve the effect of repeatability, rapidity and amplification, and high specificity

Pending Publication Date: 2020-08-07
南京实践医学检验有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] MLL (mixed lineage leukemia) gene abnormality is a very common chromosomal abnormality in acute leukemia. Most acute leukemia patients with MLL gene abnormality have a poor prognosis, which is caused by t(4;11)(q21;q23) translocation The formed MLL-AF4 fusion gene is the second most common chromosomal abnormality in acute lymphoblastic leukemia, most patients are resistant to conventional chemotherapy, and are prone to relapse even after allogeneic hematopoietic stem cell transplantation, which is an independent poor prognosis factor
[0003] Among the molecular biology methods widely used at home and abroad to detect the MLL-AF4 fusion gene, although the results of fluorescence in situ hybridization (FISH) are relatively intuitive, they can only perform qualitative detection and the operation is complicated, requiring large-scale equipment such as fluorescence microscopes and The diagnostic opinions of pathologists have strict requirements on testing institutions, which is not conducive to the rapid detection of clinical samples
Traditional solid-phase biochip (Biochip) or gene chip technology has outstanding weaknesses such as poor repeatability, insufficient sensitivity, cumbersome operation, and high cost

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A method for detecting MLL-AF4 fusion gene, comprising the following steps in sequence:

[0025] Step 1: Acquisition of sample DNA: Process the sample with reagents for extracting nucleic acid to obtain the DNA of the sample to be tested. The sample is human and animal tissue, fresh plant tissue or blood;

[0026] Step 2: Configuration of PCR reaction solution: divided into three tubes of positive control, sample and negative control, the reaction system in each tube is 20 μL, the sample DNA in the positive control tube is MLL-AF4 fusion gene solution, and the concentration is 500 μL copies / μL, sample DNA in negative control tube is ddH 2 O, and the primers used to amplify the MLL-AF4 fusion gene are:

[0027] F: 5'-CCGCCCAAGTATCCCTGTAAAAC-3';

[0028] R: 3'-TCGTTATGTATGCGGATCATGTCG-5'; the concentration of the primer sequence is 700-800nM,

[0029] The composition and components of the PCR reaction solution are DNA polymerase (2×Taq Mix) 14 μL, primer sequence F1 μL...

Embodiment 2

[0036] A method for detecting MLL-AF4 fusion gene, comprising the following steps in sequence:

[0037] Step 1: Acquisition of sample DNA: Process the sample with reagents for extracting nucleic acid to obtain the DNA of the sample to be tested. The sample is human and animal tissue, fresh plant tissue or blood;

[0038] Step 2: Configuration of PCR reaction solution: divided into three tubes of positive control, sample and negative control, the reaction system in each tube is 20 μL, the sample DNA in the positive control tube is MLL-AF4 fusion gene solution, and the concentration is 500 μL copies / μL, sample DNA in negative control tube is ddH 2 O, and the primers used to amplify the MLL-AF4 fusion gene are:

[0039] F: 5'-CCGCCCAAGTATCCCTGTAAAAC-3';

[0040] R: 3'-TCGTTATGTATGCGGATCATGTCG-5'; the concentration of the primer sequence is 700-800nM,

[0041] The composition and components of the PCR reaction solution are DNA polymerase (2×Taq Mix) 15 μL, primer sequence F1 μL...

Embodiment 3

[0048] A method for detecting MLL-AF4 fusion gene, comprising the following steps in sequence:

[0049] Step 1: Acquisition of sample DNA: Process the sample with reagents for extracting nucleic acid to obtain the DNA of the sample to be tested. The sample is human and animal tissue, fresh plant tissue or blood;

[0050] Step 2: Configuration of PCR reaction solution: divided into three tubes of positive control, sample and negative control, the reaction system in each tube is 20 μL, the sample DNA in the positive control tube is MLL-AF4 fusion gene solution, and the concentration is 500 μL copies / μL, sample DNA in negative control tube is ddH 2 O, and the primers used to amplify the MLL-AF4 fusion gene are:

[0051] F: 5'-CCGCCCAAGTATCCCTGTAAAAC-3';

[0052] R: 3'-TCGTTATGTATGCGGATCATGTCG-5'; the concentration of the primer sequence is 700-800nM,

[0053]The composition and components of the PCR reaction solution are DNA polymerase (2×Taq Mix) 16 μL, primer sequence F1 μL,...

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PUM

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Abstract

The invention discloses a method for detecting an MLL-AF4 fusion gene. The method sequentially comprises the following steps: 1, acquiring sample DNA, 2, preparing a PCR reaction liquid, wherein a primer used for amplifying the MLL-AF4 fusion gene is F: 5'-CCGCCCAAGTATCCCTGTAAAC-3', and a primer used for amplifying the MLL-AF4 fusion gene is R: 3'-TCGTTATGTATGCGGATCATGTCG-5', 3, carrying out target strip amplification by taking the DNA of a to-be-detected sample as a template, and amplifying a required DNA fragment through the designed primers, 4, carrying out target DNA recovery by carrying out 1% agarose gel electrophoresis on the PCR reaction solution, and recovering a target strip, 5, purifying the DNA fragments obtained in the step 1, 6, carrying out conversion on the purified DNA, and 7, carrying out sequencing, and judging whether the fusion gene is the MLL-AF4 fusion gene or not. According to the method for detecting the MLL-AF4 fusion gene, primers suitable for PCR amplification are designed, high specificity, accuracy and sensitivity are achieved in the detection process, a target band is amplified more rapidly and efficiently, and repeatability is achieved.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for detecting MLL-AF4 fusion gene. Background technique [0002] MLL (mixed lineage leukemia) gene abnormality is a very common chromosomal abnormality in acute leukemia. Most acute leukemia patients with MLL gene abnormality have a poor prognosis, which is caused by t(4;11)(q21;q23) translocation The formed MLL-AF4 fusion gene is the second most common chromosomal abnormality in acute lymphoblastic leukemia. Most patients are resistant to conventional chemotherapy, and they are prone to relapse even after allogeneic hematopoietic stem cell transplantation, which is an independent poor prognosis factor. [0003] Among the molecular biology methods widely used at home and abroad to detect the MLL-AF4 fusion gene, although the results of fluorescence in situ hybridization (FISH) are relatively intuitive, they can only perform qualitative detection ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2531/113C12Q2565/125C12Q2545/113
Inventor 张鹏唐春花邢宽何志健谢珍倪海钰周权何贵伦安雪茹李平江晓琴
Owner 南京实践医学检验有限公司
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