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Detection method for leukemia fusion genes, primers, probes and gene chip thereof

A fusion gene and gene chip technology, applied in the field of specific probes, multiple RT-PCR combined with gene chips to detect leukemia fusion genes, primers and gene chips, can solve problems such as unfavorable clinical screening

Active Publication Date: 2014-12-03
SHANGHAI BIOCHIP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the limitations of the main foreign chips are also quite obvious. They only target one of the fusion forms of a few types of leukemia, and the part involving PCR uses multi-tube nested PCR, which is not conducive to large-scale clinical screening.

Method used

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  • Detection method for leukemia fusion genes, primers, probes and gene chip thereof
  • Detection method for leukemia fusion genes, primers, probes and gene chip thereof
  • Detection method for leukemia fusion genes, primers, probes and gene chip thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Detection of leukemia fusion gene in K-562 cell line by multiplex RT-PCR combined with gene chip

[0025] 1. Total Cell RNA Extraction

[0026] Take the K-562 cell line, use the method of Trizol, and extract the total RNA of the cells according to the instructions of Trizol of Invitrogen Company. The specific steps of extraction are as follows:

[0027] (1) Take 400 μl of the specimen stored in Trizol, add 400 μl of chloroform, mix well, and let stand at room temperature for 5 minutes;

[0028] (2) Centrifuge at 15,000 rpm for 10 minutes, and absorb the colorless supernatant after centrifugation;

[0029] (3) Add an equal volume of pre-cooled isopropanol to the above supernatant, mix it upside down and place it at room temperature for 10 minutes;

[0030] (4) Centrifuge at 15,000 rpm for 10 minutes, and discard the supernatant;

[0031] (5) Add 300 μl pre-cooled 70% ethanol to the above centrifuge tube, centrifuge at 15,000 rpm for 5 minutes, discard the s...

Embodiment 2

[0060] Embodiment 2 multiplex RT-PCR detection result

[0061] Take KASUMI-1, NB4, ME-1, THP-1, REH and K-562 leukemia cell lines, and t(15;17)PML-RARA S type, t(4;11)MLL-AF4, t(11 ;19)MLL-ENL, t(11;19)MLL-ELL, t(6;11)MLL-AF6, t(10;11)MLL-AF10, t(1;19)E2A-PBX1, t(9 22) For BCR-ABL p190 positive samples, perform the extraction of total cellular RNA, specific reverse transcription reaction and multiplex PCR amplification test according to Step 1 to Step 3 in Example 1. After the end of the test, get 3 μl of PCR product and detect it with 2% agarose gel, the results of electrophoresis are shown in image 3 .

[0062] image 3 KASUMI-1, NB4, ME-1, THP-1, REH, and K-562 leukemia cell lines are shown, along with t(15;17)PML-RARA type S, t(4;11)MLL-AF4, t (11;19)MLL-ENL, t(11;19)MLL-ELL, t(6;11)MLL-AF6, t(10;11)MLL-AF10, t(1;19)E2A-PBX1, t (9;22) Multiplex RT-PCR results of BCR-ABL p190 positive samples. The band at 328bp is the internal reference gene GUS gene, and the other b...

Embodiment 3

[0063] Embodiment 3 Sensitivity detection

[0064] Taking KASUMI-1 cells as an example, the sensitivity experiment was carried out. Starting from 2 μg of initial RNA, carry out 10-fold serial dilution with RNA of HL-60 (negative leukemia cells), and the lowest dilution is 10 -4 . Multiplex RT-PCR combined with gene chip detection to determine the sensitivity of KASUMI-1 cells, see the results Figure 4 .

[0065] Figure 4 Gel electrophoresis and chip hybridization of different dilution gradients of KASUMI-1 are shown in . It can be seen from the results of gel electrophoresis and chip hybridization that the detection sensitivity of KASUMI-1 is within 10 -3 , that is, as long as there is one cell with the AML1-ETO fusion gene in 1000 cells, it can be obtained by the method of the present invention.

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Abstract

The invention discloses a method for detection of leukemia fusion genes through combination of multiplex RT-PCR and a gene chip. The method includes the steps of: 1) extracting total RNA of a sample cell; 2) subjecting RNA to specific reverse transcription to obtain cDNA; 3) conducting multiple RT-PCR amplification on the cDNA; and 4) detecting the PCR amplification product by the gene chip containing specific probes for detecting leukemia fusion genes. The invention also discloses the probes, the primers and the gene chip adopted by the method. A tube nested multiplex PCR is employed to amplify specific fusion gene fragments possibly contained in the sample, and then the fusion gene detection result can be obtained through gene chip hybridization and chip scanning image analysis. In this way, simultaneous detection of 27 or more different fusion forms of specific fragments of 15 leukemia fusion genes can be realized, thus greatly improving the detection efficiency of leukemia fusion genes, and being in favor of large-scale clinical screening.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method for detecting leukemia fusion genes by multiple RT-PCR combined with a gene chip, and specific probes, primers and gene chips used in the method. Background technique [0002] Leukemia is a malignant tumor of the hematopoietic system, with an annual incidence of about 2.76 per 100,000 people, accounting for 5% of the total number of cancers. It ranks first in the mortality rate of malignant tumors in children and adults under the age of 35. The occurrence and development of leukemia is a complex process, which often involves the changes of multiple genes, including the mutation or deletion of normal genes, the abnormal amplification and expression of oncogenes, and the synergistic effect of multiple genes, combined with the pleiotropic effects of the genes themselves and the immune system. Factors that ultimately determine whether a tumor phenotype is expressed or...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6837C12Q1/6848C12Q2565/501C12Q2531/113C12Q2537/143
Inventor 熊斐斐张春秀张庆华熊慧
Owner SHANGHAI BIOCHIP
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