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Primers, kit and method for qualitatively detecting leukaemia fusion genes

A technology for detecting primers and fusion genes, which is applied in the field of genetic engineering and can solve the problems of increasing the probability of multiple pairs of primers interacting and forming primer-dimers, failing to amplify specific target products, and reducing the specificity and efficiency of PCR reactions. , to achieve the effect of saving reagent consumption, reducing detection cost, and improving specificity and efficiency

Active Publication Date: 2016-08-10
SHANGHAI TISSUEBANK BIOTECH +3
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] However, multiple nested PCR technology needs to add multiple pairs of primers in the same PCR reaction system, which increases the probability of multiple pairs of primers interacting in the reaction system and forming primer-dimers, thereby reducing the specificity of the PCR reaction. and efficiency, even the specific target product cannot be amplified
In addition, as people's research on leukemia continues to deepen, more and more leukemia fusion genes have been discovered, and the current methods for detecting leukemia fusion genes are far from meeting people's needs for simultaneous detection of most fusion genes. A rapid, effective and easily standardized method for the simultaneous qualitative detection of the vast majority of leukemia fusion genes

Method used

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  • Primers, kit and method for qualitatively detecting leukaemia fusion genes
  • Primers, kit and method for qualitatively detecting leukaemia fusion genes
  • Primers, kit and method for qualitatively detecting leukaemia fusion genes

Examples

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Effect test

Embodiment 1

[0039] Embodiment 1: Utilize fusion gene positive cell line and negative cell line to detect the specificity of detection system of the present invention

[0040] In this example, 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 fusion gene, the Kasumi cell line containing the AML1-ETO fusion gene, and the HL60 negative cell line without the fusion gene. Among them, K562 cell line, Kasumi cell line, and HL60 cell line were all purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0041] The specific detection method of the present embodiment is as follows:

[0042] 1. Cell culture

[0043] 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.

[0044] 2. Nucleic acid extraction

[0045] It is recommended to use TIANGEN RNAprep Pure Blood Kit, follo...

Embodiment 2

[0059] Embodiment 2: Utilize fusion gene positive cell line and negative cell line to detect the sensitivity of detection system of the present invention

[0060] In this embodiment, the sensitivity of the detection system of the present invention is also verified by determining the K562 positive cell line containing the BCR-ABL1 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:

[0061] 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 nested RT-PCR detection.

[0062] Usin...

Embodiment 3

[0064] Example 3: Detection of clinical samples using the detection system of the present invention

[0065] In this example, 150 peripheral blood or bone marrow blood samples were collected from clinical leukemia patients, and the qualitative detection of 43 fusion genes was carried out according to the detection system of the present invention. Among them, 25 cases of clinical samples were detected as fusion gene positive by multiple nested RT-PCR of the present invention, and 125 cases were fusion gene negative.

[0066] Using the multiplex nested RT-PCR detection system of the present invention to detect clinical samples, the operation steps include leukocyte RNA extraction from blood samples, RNA reverse transcription into cDNA, first round reaction of nested PCR, second round reaction of nested PCR, separation PCR Reaction products, agarose gel electrophoresis. The specific operation method is the same as that of Embodiment 1 of the present invention. For the results o...

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Abstract

The invention belongs to the technical field of gene engineering, and discloses a primer combination for detecting leukaemia fusion genes, a kit containing the primer combination, and a multiplex nested RT-PCR (reverse transcription-polymerase chain reaction) method for performing leukaemia fusion gene detection by using the primer combination or kit. The method is based on a multiplex nested RT-PCR technique, and thus, is simple and quick and has high sensitivity. Besides, the reasonable primer combination is utilized to effectively avoid interactions among multiple primer pairs, thereby reducing the detection errors. The detection method can be utilized to comprehensively perform qualitative detection on 43 leukaemia fusion genes, thereby saving the reagent consumption and lowering the detection cost. The detection method has wide detection range, and is suitable for detecting mass samples in clinic.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to primers, 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 myeloid l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6848C12Q1/6886C12Q2600/16C12Q2537/143C12Q2531/113C12Q2521/107
Inventor 郑仲征杜金伟张鹏王宁娟潘捷杜可明
Owner SHANGHAI TISSUEBANK BIOTECH
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