PML/RAR alpha fusion gene detection kit and detection method thereof

A detection kit and fusion gene technology, which are applied in the field of molecular biology, can solve the problems of inability to type PML-RARα fusion genotype, weak signals are difficult to be detected, and affect hybridization specificity, etc., so as to improve detection sensitivity and detection efficiency. Guaranteed result specificity and sensitivity, the effect of strong specificity

Active Publication Date: 2017-08-22
SUREXAM BIO TECH
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the FISH detection method has been widely used and improved, but still has the following defects: (1) There are many repetitive sequences in the probe prepared with BAC as the template, which affects the specificity of hybridization and causes high background fluorescence (2) The detection steps are complicated, the probe sequence is too long, and it often takes 12-16 hours for the probe to hybridize with the sample to ensure sufficient hybridization, which is time-consuming and laborious; (3) Some optimized probe sequences are too short, resulting in reduced detection specificity and The signal is weak and difficult to detect; (4) Due to the short distance between the breakpoints, the existing FISH products cannot type the PML-RARα fusion gene

Method used

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  • PML/RAR alpha fusion gene detection kit and detection method thereof
  • PML/RAR alpha fusion gene detection kit and detection method thereof
  • PML/RAR alpha fusion gene detection kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 kit composition

[0037] The PML-RARα fusion gene detection kit described in this embodiment mainly includes:

[0038] 1. Fluorescent dye-labeled probes

[0039] The probes include a first group of probes directed at the upstream gene sequence of the L-type break hotspot of the PML gene and a second group of probes directed at the entire gene sequence of RARα. The probe is obtained by using human peripheral blood cell DNA as a template, and using 30 pairs of specific primer pairs to carry out PCR amplification reaction, and its preparation method is as follows:

[0040] 1. Design of amplification primers

[0041] The PML gene located at 15q22 and the RARα gene located at 17q21 were translocated to form a PML-RARα fusion gene. Because the breakpoints of the PML gene are different, and the breakpoint of RARα is constant (intron 2), different PML-RARα fusion gene isomers can be produced, such as L-type fusion gene, S-type fusion gene and V-type fusion gene....

Embodiment 2

[0062] Example 2 Using the kit of Example 1 to detect clinical samples

[0063] 1. Sample pretreatment

[0064] 1. Collect 1-1.5ml of peripheral blood from the patient with a sodium heparin anticoagulant tube, and centrifuge at 2000rpm for 5min;

[0065] 2. Discard the supernatant, add 5-10ml 0.075M KCl solution, pipette evenly, and treat with hypotonicity at 37°C for 30min;

[0066] 3. Add 1ml of fresh fixative solution (methanol / glacial acetic acid 3:1), pipette evenly, and let stand for 5 minutes;

[0067] 4. Centrifuge at 2000rpm for 5min, discard the supernatant;

[0068] 5. Add 10ml of fresh fixative solution (methanol / glacial acetic acid 3:1), pipette evenly, and let stand for 10min;

[0069] 6. Centrifuge at 2000rpm for 5min, discard the supernatant;

[0070] 7. Add 10ml of fresh fixative solution (methanol / glacial acetic acid 3:1), pipette evenly, centrifuge at 2000rpm for 5min, and discard the supernatant;

[0071] 8. Repeat step 7 twice, and finally add 10ul of...

Embodiment 3

[0098] Embodiment 3 The influence of the length of hybridization on the detection effect of the kit

[0099] 1. Hybridization time

[0100] The design of the probe length and dye labeling method of the kit of the present invention is the optimal solution obtained by the inventor after a large number of experiments, comparison and statistical analysis of the experimental results, which can achieve the optimal detection specificity and hybridization time. Balance can not only ensure the specificity and sensitivity of the results, but also shorten the hybridization time and improve the detection efficiency.

[0101] In order to verify the influence of hybridization duration on the detection results of the present invention, this example set up 4 experimental groups with different hybridization durations, see Table 4 for details, and the detection probes and reagents used were consistent with the kit in Example 1.

[0102] Table 4 Hybridization time

[0103] group Ex...

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Abstract

The invention discloses a PML-RAR alpha fusion gene detection kit and a detection method thereof. The kit comprises a first group of probes for detecting an upstream gene sequence of an L-type breakage hot spot of a PML gene and a second group of probes for detecting all gene sequences of a RAR alpha gene. Each one of probes is labeled a dye. The dyes labeling the two groups of probes are in different colors. The two groups of probes are amplification products obtained by amplification based on a human epigenome DNA as a template and amplification primers. The probes have strong specificity and low background noise and realizes the optimal balance between the detection specificity and hybridization time. The PML-RAR alpha fusion gene detection kit guarantees result specificity and sensitivity, shortens hybridization time, has detection time of 7-8h and improves detection efficiency.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a PML / RARα fusion gene detection kit and detection method. Background technique [0002] Acute promyelocytic leukemia (acute promyelocytic leukemia, APL) is a special type of acute myeloid leukemia, which is designated as acute myeloid leukemia type M3 by the FAB collaboration group, and is characterized by a large number of leukemia cells in the bone marrow and other hematopoietic tissues Unrestricted proliferation, and into the peripheral blood, while the production of normal blood cells is significantly inhibited, the disease ranks first in young malignant diseases, the etiology is still not completely clear. 95% of APL patients have t(15; 17) chromosomal abnormality, and the formed PML-RARα fusion gene is its molecular marker. [0003] The PML gene located at 15q22 and the RARα gene located at 17q21 were translocated to form a PML-RARα ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6841C12Q2563/107
Inventor 朱蓉吴诗扬廖传荣
Owner SUREXAM BIO TECH
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