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TaqMan MGB probe real-time fluorescence PCR detection kit for leukemia fusion genes

A fusion gene, real-time fluorescence technology, applied in DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of low specificity and sensitivity, time-consuming and labor-intensive, etc., achieve accurate detection results, reduce background signal interference, improve Effects of Differences in Tm Values

Active Publication Date: 2013-07-24
北京旌准医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0042] The present invention provides a TaqMan-MGB real-time fluorescent PCR detection method for leukemia fusion genes to solve the problems of low specificity and sensitivity, time-consuming and labor-intensive detection methods for leukemia fusion genes

Method used

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  • TaqMan MGB probe real-time fluorescence PCR detection kit for leukemia fusion genes
  • TaqMan MGB probe real-time fluorescence PCR detection kit for leukemia fusion genes
  • TaqMan MGB probe real-time fluorescence PCR detection kit for leukemia fusion genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1. Design and screening of primers and TaqMan-MGB probes for real-time fluorescent PCR detection of leukemia fusion genes

[0067] According to leukemia fusion genes AML1-ETO, BCR-ABL190, BCR-ABL210, CBFb-MYH11(A), CBFb-MYH11(D), CBFb-MYH11(E), E2A-PBX1, MLL-AF4, PML-RARa(bcr1 ), PML-RARa(bcr2), PML-RARa(bcr3), SIL-TAL1 and TEL-AML1 conserved regions designed primers and probes for TaqMan-MGB real-time fluorescent PCR detection, wherein 5 of the probe The 'end is fluorescently labeled with a reporter luminescent group FAM, the 3' end is labeled with a non-fluorescent quencher group NFQ, and the probe is also connected with a MGB (Minor Groove Binder) modification group. The screening principle of primers / probes is: select primers and probes with higher PCR amplification efficiency and probe binding efficiency in the target conserved region as candidate primers and probes. After screening, primers and TaqMan-MGB probes of the present invention were obtained, and...

Embodiment 2

[0110] Embodiment 2, TaqMan-MGB probe real-time fluorescent PCR detection of leukemia fusion gene

[0111] Detect 13 clinical samples with the TaqMan-MGB probe real-time fluorescence quantitative PCR detection method of the leukemia fusion gene of the present invention, the specific method comprises the following steps:

[0112] 1) Extract 13 confirmed leukemia patients (known to carry AML1-ETO, BCR-ABL190, BCR-ABL210, CBFb-MYH11(A), CBFb-MYH11(D), CBFb-MYH11(E), E2A-PBX1 , MLL-AF4, PML-RARa(bcr1), PML-RARa(bcr2), PML-RARa(bcr3), SIL-TAL1 and TEL-AML1 genes) and a peripheral blood sample from a healthy subject (control) (bone marrow or tissue is also acceptable) and 1 part of peripheral blood RNA of healthy subjects.

[0113] 2) Reverse transcribe the peripheral blood RNA of the subject extracted in step 1) into cDNA, the 20ul reverse transcription system is (all reagents are purchased from Promega): RNA 4ul (400ng), reverse transcription master mix 5.75 ul (MMLV 0.5 ul, MML...

Embodiment 3

[0117] Embodiment 3, the preparation of the TaqMan-MGB probe real-time fluorescent PCR detection kit of leukemia fusion gene

[0118] The reaction solution (premix solution) used for real-time fluorescent PCR detection of leukemia fusion gene by TaqMan-MGB probe is composed of the following components: upstream primer 1-3μL (2.5-7.5uM), downstream primer 1-3μL ( 2.5-7.5uM), 1 μL of TaqMan-MGB probe (1.25uM), 10 μL of Universal Master Mix for PCR (2×, purchased from ABI Company), and make up to 18 μL with nuclease-free water. Specifically, the test kit of the present invention includes the following components:

[0119] (1) Reverse transcription system, the components are as follows:

[0120] Reagent

company

Item No.

Volume (20ul*20)

MMLV

Promega

M1705

0.5ul*20

MMLV reaction buffer

Promega

M1705

4ul*20

RNase inhibitor

Promega

N2615

0.25ul*20

nuclease free water

QIAGEN

129112

10.25...

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Abstract

The invention discloses the TaqMan MGB probe real-time fluorescence PCR detection method for leukemia fusion genes, and special primers, probes and kit used by the same. In the method disclosed by the invention, leukemia fusion genes (AML1-ETO, BCR-ABL190, BCR-ABL210, CBFb-MYH11(A), CBFb-MYH11(D), CBFb-MYH11(E), E2A-PBX1, MLL-AF4, PML-RARa(bcr1), PML-RARa(bcr2), PML-RARa(bcr3), SIL-TAL1 and TEL-AML1) in a sample to be detected are used as detection objects, and the accuracy and precision are both high. When the kit disclosed by the invention is used for detecting the 8 leukemia fusion genes in a leukemia patient, the specificity is as high as 100 percent; and the sensitivity is as high as 0.01 percent, namely 1 leukemia cell is detected in 10,000 cells. The kit can provide a direct theoretical basis for the diagnosis of leukemia and can provide a powerful means for treatment effect observation, determination after prediction, detection of minimal residual disease and the like; and theoperation is simple and convenient, the stability is high, and the kit has prefunding clinic significance and popularization property. The detection method and the kit, which are disclosed by the invention, can be used for qualitative detection of leukemia fusion genes in various clinic samples (marrow, peripheral blood or tissue) and have bright application prospects.

Description

technical field [0001] The invention relates to molecular biological detection of genes in the field of biotechnology, in particular to a TaqMan-MGB probe real-time fluorescent PCR detection kit for leukemia fusion genes and its special primers and probes. Background technique [0002] Human beings have recognized leukemia for more than one and a half centuries. Hematopoietic stem and progenitor cells lose the ability to further differentiate and mature due to malignant changes, making the cells stagnate in different hematopoietic stages, resulting in a group of heterogeneous hematopoietic malignancies tumor. Leukemia is the most common malignant tumor in children and adolescents, occupying the first place among malignant tumors in children and adolescents; the annual morbidity and mortality rate in my country is about 3-4 / 100,000, ranking sixth among all kinds of tumors. [0003] t(8;21)(q22,q22) is mainly seen in patients with acute myeloid leukemia (AML), accounting for ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 徐玉现毛吉扬吴赓
Owner 北京旌准医疗科技有限公司
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