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Multiplex fluorescent PCR kit and method for detecting fusion genes of leukemia

A fusion gene and leukemia technology, applied in biochemical equipment and methods, recombinant DNA technology, and microbial assay/inspection, etc., can solve problems such as low detection sensitivity, cumbersome detection procedures, and inappropriate early diagnosis, and achieve high detection sensitivity. , easy operation, easy results

Active Publication Date: 2017-03-29
XUZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection procedure of this method is cumbersome, and a patient needs multiple PCR reactions and gel electrophoresis for identification; the detection sensitivity is low, and the price is generally more than 1,000 yuan, which is not suitable for early diagnosis

Method used

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  • Multiplex fluorescent PCR kit and method for detecting fusion genes of leukemia
  • Multiplex fluorescent PCR kit and method for detecting fusion genes of leukemia
  • Multiplex fluorescent PCR kit and method for detecting fusion genes of leukemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1. Establishment of primer pairs and methods for detecting leukemia

[0070] 1. Primer pair for detection of leukemia

[0071] Based on 11 leukemia fusion genes: E2A-PBX1, MLL-AF4, TEL-AML1, BCR-ABL p210, BCR-ABLp190, SIL-TAL, PML-RARA L type, PML-RARA S type, AML1-ETO, CBFB- For MYH11A type and CBFB-MYH11DE type, design the primer pairs shown in Table 1 below.

[0072] Table 1 is the primer pair for detecting leukemia

[0073]

[0074]

[0075]

[0076]

[0077] 2. Kit or system for detecting leukemia

[0078] Multiplex fluorescent PCR reaction system, including 23 pairs of primers in Table 1, Taq DNA polymerase, dNTP, SYBRGreen, MgCl 2 , PCR buffer and ultrapure water.

[0079] The multiple fluorescent PCR reaction system of the present invention is 50 μL, including Taq DNA polymerase 1U, dNTPs final concentration 400 μM, MgCl 2 The final concentration is 3mM, the final concentration of SYBR Green is 2μM, the cDNA of the sample to be detected ...

Embodiment 2

[0092] Embodiment 2, the application of the primer pair that detects leukemia

[0093] The kit and method of Example 1 were used to detect clinical samples from healthy people and leukemia patients.

[0094] In this example, 20 peripheral blood samples of healthy people and 20 peripheral blood samples of fusion gene-positive leukemia patients were collected (patients were informed).

[0095] 1. Acquisition of sample cDNA

[0096] (1) Peripheral blood samples should be anticoagulated with EDTA

[0097] Add 25ml of erythrocyte lysate to a 50ml centrifuge tube. Mix the isolated peripheral blood sample upside down, pour it into a 50ml centrifuge tube, place it on a roller shaker for 10 minutes, and centrifuge at 1500 rpm for 5 minutes. Add 1ml of phosphate buffer and blow it up, transfer to a 1.5ml centrifuge tube, centrifuge at 1600 for 3 minutes, then add phosphate buffer, divide into tubes according to the number of cells, about 10 per tube 7 cells. Centrifuge at 1600 rpm ...

Embodiment 3

[0106] Embodiment 3, kit and method sensitivity detection of the present invention

[0107] 1. Obtaining the template

[0108] Using the constructed BCR-ABL p210 plasmid standard as a template (recorded in the following literature: "Leukemia. Lymphoma", 2007, 16 (03): 161-164), the plasmid was diluted to 10 5 、10 4 、10 3 、10 2 、10 1 copies / μL.

[0109] 2. PCR amplification

[0110] 50 μL of PCR reaction system: 25 μL of Taq enzyme master mix (purchased from ABI Company) (containing Taq enzyme, dNTPs, MgCl 2 , SYBR Green), plasmid standard 1 μL, primer mixture 5 μL (to make the final concentration of the fusion gene primer in the PCR reaction system is 0.06 μM, and the final concentration of the universal primer in the PCR reaction system is 0.3 μM), ultrapure Make up to 50 μL with water. Put the prepared PCR reaction system into a 96-well PCR plate, cover with film, seal, and centrifuge to mix.

[0111] The multiplex fluorescent PCR reaction conditions are: 95°C for 3...

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Abstract

The invention discloses a multiplex fluorescent PCR kit and a method for detecting fusion genes of leukemia. The invention provides a complete set of primers for detecting the fusion genes of the leukemia. The complete set of primers consists of primer pairs 1-23; and nucleotide sequences of the primer pairs 1-23 are sequences 1-46 respectively. The kit and the detection method provided by the invention are low in cost, are simple and convenient in operation, are high in detection sensitivity and easy in result interpretation, and are suitable for detection of large-batch samples in daily health examination.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a multiple fluorescent PCR kit and method for detecting leukemia fusion genes. Background technique [0002] Leukemia is a kind of clonal malignant disease with abnormal hematopoietic stem cells, and it is one of the malignant cancers with high incidence in children and young people. Leukemia has multiple subtypes with complex and variable genetic characteristics. With the development of molecular biology techniques, it has been confirmed that many leukemia patients have chromosomal variations, including chromosomal translocations, inversions, and deletions. Chromosomal variation in leukemia patients will produce new fusion genes, and these fusion genes are key factors in the pathogenesis of leukemia. The fusion gene can be used as a molecular marker for the diagnosis and typing of leukemia. For example, BCR-ABL p210 fusion gene is the key factor of chronic myeloid leukemia, more ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/16C12Q2537/143C12Q2563/107
Inventor 牛铭山徐开林姚瑶刘雪娇齐家磊沈洋灵杨雪
Owner XUZHOU MEDICAL UNIV
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