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

A detection kit and gene fusion technology, applied in the field of molecular biology, can solve the problems of complicated detection steps, too long probe sequences, difficult to be detected, etc., so as to improve detection sensitivity and detection efficiency, and ensure the specificity and sensitivity of results. , the effect of shortening the time required for full hybridization

Active Publication Date: 2017-08-22
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
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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

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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 for PML gene and a second group of probes for RARα gene. The probe is obtained by using human genomic DNA as a template and using 40 pairs of specific primers to carry out PCR amplification reaction, and its preparation method is as follows:

[0040] 1. Design of amplification primers

[0041] The PML-RARα fusion gene is formed by the mutual translocation of the PML gene located at 15q22 and the RARα gene located at 17q21. Since the PML gene and the RARα gene are relatively small in length, after excluding repetitive sequences and highly variable sequences, the area available for probe preparation is limited. Therefore, the preparation of the probe of the present invention selects the upstream and downstream sequences of 200 kb and 200kb sequence of up...

Embodiment 2

[0064] Example 2 Using the kit of Example 1 to detect clinical samples

[0065] 1. Sample pretreatment

[0066] 1. Collect 1-1.5ml of peripheral blood from the patient with a sodium heparin anticoagulant tube, and centrifuge at 2000rpm for 5min;

[0067] 2. Discard the supernatant, add 5-10ml 0.075M KCl solution, pipette evenly, and treat with hypotonicity at 37°C for 30min;

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

[0069] 4. Centrifuge at 2000rpm for 5min, discard the supernatant;

[0070] 5. Add 10ml of fresh fixative solution (methanol / glacial acetic acid 3:1), pipette evenly, and let stand for 10min;

[0071] 6. Centrifuge at 2000rpm for 5min, discard the supernatant;

[0072] 7. Add 10ml of fresh fixative solution (methanol / glacial acetic acid 3:1), pipette evenly, centrifuge at 2000rpm for 5min, and discard the supernatant;

[0073] 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 upstream and downstream sequences of a PML gene and a second group of probes for detecting upstream and downstream sequences of a RAR alpha gene. Each one of the probes is labeled with 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 genomic DNA as a template and amplification primers. The length of the probes is designed according to a lot of experiments. Through experiment result comparison and statistic analysis, the optimal length is obtained and the optimal balance between the detection specificity and hybridization time is realized. 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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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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