Probe set, kit and method for rapid detection of BCR/ABL gene fusion

A gene fusion and probe set technology, applied in the field of molecular biology, can solve the problems of unfavorable signal confirmation and statistics, reduction of fluorescence signal intensity, divergence of fluorescence signal points, etc., to reduce non-specific hybridization, avoid non-specific hybridization, speed quick effect

Inactive Publication Date: 2017-10-03
SUZHOU ZHONGSHENG DAMAIDI MOLECULE DIAGNOSTICS TECH
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Problems solved by technology

[0003] Therefore, BCR / ABL gene fusion has been listed as a must-test item in clinical leukemia. At present, the common methods for clinically detecting BCR / ABL gene fusion include PCR and fluorescence in situ hybridization (FISH): PCR has high detection sensitivity, but it can only be detected with It is limited to the detection of known transcripts and can only detect a small range of gene sequences. When the gene sequence is mutated, it is easy to produce false negative results; fluorescence in situ hybridization (FISH) is a method for detecting BCR / ABL gene fusion gold standard
However, traditional fluorescence in situ hybridization (FISH) uses genomic probes, and the total length of the probes is more than 100Kb. Due to the extremely long length of the probes and the large coverage, non-specific hybridization inevitably occurs in some sequences, resulting in background bias. High; at the same time, too long probes often lead to divergence of fluorescent signal points, which is not conducive to signal confirmation and statistics
Due to the limited fluorescence intensity per unit length of traditional fluorescent in situ hybridization (FISH) probes, the method of reducing the length of the probe to solve the above problems will reduce the fluorescence signal intensity, which is not conducive to detection

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  • Probe set, kit and method for rapid detection of BCR/ABL gene fusion

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Effect test

Embodiment 1

[0041] Example 1: Design and synthesis of three kinds of probes: contact probe, amplification probe and fluorescent probe

[0042] (1) Through sequence comparison and advanced structural analysis, the sequences of 30 bp on the BCR gene and the ABL gene were respectively selected as the detection target site 1 and the detection target site 2, and their base sequences were respectively:

[0043] Target site 1: 5'-GCTACCGTTTCCAGTAGTTATTCCCCCTCCA-3'

[0044] Target site 2: 5'-TCCCCCTCCATCAGGCAGTTTCCCAGACAT-3'

[0045] (2) According to the sequence of the target site, design a complementary probe sequence, and add 20 groups of 20-base repeat sequences at its 3' end to form contact probe 1 and contact probe 2, and their base sequences are respectively:

[0046] Contact probe 1:

[0047] 5'-TGGAGGGGGATAACTACTGGAAACGGTAGC (GTATJCGCJCTGFTATJCCG) 20 -3'

[0048] Contact Probe 2:

[0049] 5'-ATGTCTGGGAAACTGCCTGATGGAGGGGGA(AGTFAJCGCFGTAFCAAJTJ) 20 -3'

[0050] The repetitive sequen...

Embodiment 2

[0063] Embodiment 2: traditional FISH probe and probe set of the present invention detect BCR / ABL gene fusion

[0064] A. Traditional FISH probe detection

[0065] A method for detecting BCR / ABL gene fusion using traditional FISH probes, comprising the following steps:

[0066] (1) Take 10 μL of sample and drop it on the glass slide, and dry it at 56°C to fix the sample on the glass slide;

[0067] (2) Dewax the slides by immersing them in xylene for 2 times at room temperature, each time for 10 minutes, then immerse them in 100% ethanol for 5 minutes, then immerse them in 100% ethanol, 85% ethanol and 70% ethanol for 2 minutes each. water, immerse in purified water at 90°C for 20-30min, take out the glass slide for observation every 5min, to prevent the sample from falling off, take out the glass slide, and immerse it in 70% ethanol, 85% ethanol and 100% ethanol in sequence at room temperature Dehydrate in ethanol for 2 minutes each, and dry naturally;

[0068] (3) Add 3 μ...

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Abstract

The invention discloses a method for rapid detection of BCR/ABL gene fusion by using fluorescence in-situ hybridization technology. The method is characterized in that detection of each target site needs three kinds of probes, i.e., contact probes, amplification probes and fluorescence probes; the 5' terminals of the contact probes specifically bind to a BCR gene (or an ABL gene), and the 3' terminals of the contact probes both have 20 sets of a repetitive sequence consisting of 20 bases and can specifically bind to the amplification probes; the 5' terminals of the amplification probes specifically bind to the 3' terminals of the contact probes, and the 3' terminals of the amplification probes both have 20 sets of a repetitive sequence consisting of 20 bases and can specifically bind to the fluorescence probes; the 5' terminals of the fluorescence probes are labeled by CY3 or FITC; and through synergism of the three kinds of probes, a fluorescence signal is amplified by 400 times, and thus, rapid and sensitive detection of BCR/ABL gene fusion is realized. The invention also discloses a kit and probe set for rapid detection of BCR/ABL gene fusion.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a probe set and a reagent kit for rapidly detecting BCR / ABL gene fusion in a sample by means of fluorescence in situ hybridization. Background technique [0002] Chronic myelogenous leukemia (CML) is a malignant clonal proliferative disease of the blood system that occurs in hematopoietic stem cells. Chronic myeloid leukemia is one of the malignant blood diseases that seriously endanger the health of young and middle-aged people, and it is a more common type of leukemia. The characteristic Philadelphia chromosome and its molecular marker BCR / ABL gene fusion appear in white blood cells of more than 95% of CML patients. Patients diagnosed with BCR / ABL gene fusion can be treated precisely with targeted drug tyrosine kinase inhibitors (such as Gleevec, etc.). [0003] Therefore, BCR / ABL gene fusion has been listed as a must-test item in clinical leukemia. At present, the common met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6841C12Q1/6886C12Q2600/166C12Q2600/118C12Q2600/106C12Q2563/107C12Q2525/117
Inventor 方国伟洪冉
Owner SUZHOU ZHONGSHENG DAMAIDI MOLECULE DIAGNOSTICS TECH
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