Primer, probe, kit and method for qualitatively detecting fusion genes of leukemia

A fusion gene and leukemia technology, applied in the field of genetic engineering, can solve the problems of inability to amplify specific target products, reduce the specificity and efficiency of PCR reactions, increase the interaction of multiple pairs of primers and multiple probes, and form dimers Probability and other issues, to achieve shortened detection time, high sensitivity and specificity, and reduce false positive effects

Active Publication Date: 2016-08-10
SHANGHAI TISSUEBANK BIOTECH +3
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] However, multiple fluorescent RT-PCR technology needs to add multiple pairs of primers and even multiple probes in the same PCR reaction system, which increases the interaction of multiple pairs of primers and multiple probes in the reaction system and the formation of dimerization. The probability of the body, thereby reducing the specificity and efficiency of the PCR reaction, and even the specific target product

Method used

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  • Primer, probe, kit and method for qualitatively detecting fusion genes of leukemia
  • Primer, probe, kit and method for qualitatively detecting fusion genes of leukemia
  • Primer, probe, kit and method for qualitatively detecting fusion genes of leukemia

Examples

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Embodiment 1

[0044] Embodiment 1: Utilize fusion gene positive cell line and negative cell line to detect the specificity of detection system of the present invention

[0045] In this embodiment, the feasibility and specificity of the detection method of the present invention are verified by determining the K562 positive cell line containing the BCR-ABL1 (p210) fusion gene, the Kasumi cell line of the AML1-ETO fusion gene, and the HL60 negative cell line without the fusion gene sex. Among them, K562 cell line, Kasumi cell line, and HL60 cell line were all purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0046] The specific detection method of the present embodiment is as follows:

[0047] 1. Cell culture

[0048] Cultivate K562 cell line, Kasumi cell line and HL60 cell line according to the standard operation of cell culture at 37°C, 5% CO 2 Cultured in an incubator.

[0049] 2. Nucleic acid extraction

[0050] It is recommended to use TIANGEN RNAprep Pure Blood Kit...

Embodiment 2

[0064] Example 2: Using fusion gene positive cell lines to detect the repeatability of the detection system of the present invention

[0065] In this embodiment, the repeatability of the detection system of the present invention is also verified by determining the K562 positive cell line containing the BCR-ABL1 fusion gene and the Kasumi cell line containing the AML1-ETO fusion gene.

[0066] The specific detection method is as follows:

[0067] Using the same multiple fluorescent RT-PCR reaction system and reaction conditions as in Example 1, using K562cDNA and Kasumi cDNA as templates, each sample was repeatedly detected three times, and the fluorescent PCR amplification curve for repeatability detection can be found in the appendix of the instructions. image 3 . in image 3 A is the fusion gene detection result of K562 positive cell line, image 3 B is the fusion gene detection result of Kasumi positive cell line. Such as image 3 As shown in A, the positive cell line...

Embodiment 3

[0068] Embodiment 3: Utilize fusion gene positive cell line and negative cell line to detect the sensitivity of detection system of the present invention

[0069] This embodiment also verifies the sensitivity of the detection system of the present invention by determining the K562 positive cell line containing the BCR-ABL1 (p210) fusion gene, the Kasumi cell line containing the AML1-ETO fusion gene, and the HL60 negative cell line without the fusion gene. The specific detection method is as follows:

[0070] K562 cell line (positive cell line containing BCR-ABL1 fusion gene) and Kasumi cell line (positive cell line containing AML-ETO fusion gene) were carried out with HL-60 cell line (negative cell line without fusion gene) respectively. Dilution of 100%, 10%, 1%, 1‰, 0.5‰, 0.25‰, 0.125‰ (the ratio of positive cell line to negative cell line after dilution), extract RNA with diluted mixed cell line and reverse transcribe into cDNA , for multiplex fluorescent RT-PCR detection....

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Abstract

The invention belongs to the technical field of gene engineering and discloses a prime-probe combination for detecting fusion genes of leukemia, a kit containing the prime-probe combination and a multiplex fluorescent RT-PCR method for detecting the fusion genes of the leukemia by virtue of the prime-probe combination or the kit. Based on a multiplex fluorescent RT-PCR technique, the method is simple, rapid and high in sensitivity. Besides, by virtue of the reasonable prime-probe combination, the mutual action between the two primers, the prime and the probe as well as two probes are effectively avoided, and the detection error is reduced. By virtue of the method, 45 fusion genes of the leukemia can be comprehensively and qualitatively detected, the detection time is effectively shortened, and very important detection measures are provided for the evaluation of clinical diagnosis and treatment and the prognosis of the leukemia.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to primers, probes, kits and methods for qualitative detection of leukemia fusion genes. Background technique [0002] Leukemia is the most common malignant clonal disease in children and young adults. A number of studies have shown that most leukemia patients have certain chromosomal aberrations (deletions, duplications, inversions, translocations, etc.), which are important reasons for the occurrence and development of leukemia. The aberration of chromosomal structure will lead to the structural variation of proto-oncogene and tumor suppressor gene, make oncogene and proto-oncogene mutation activation, tumor suppressor gene deletion or inactivation, produce new fusion gene and encode fusion protein. Therefore, different fusion genes have become the specific markers of molecular biology of different types of leukemia. For example, the BCR-ABL1 fusion gene in chronic m...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/16C12Q2537/143C12Q2563/107
Inventor 郑仲征杜金伟张鹏王莉萍潘捷杜可明
Owner SHANGHAI TISSUEBANK BIOTECH
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