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BCR/ABL gene fusion and ASS gene deletion detection kit

A detection kit and gene deletion technology, applied in the field of molecular biology, can solve the problems of weak signal, difficult to be detected, affecting hybridization specificity, and detection specificity reduction, so as to improve detection sensitivity, strong specificity, and low background noise Effect

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

AI Technical Summary

Problems solved by technology

Although the current FISH detection method has been widely used and improved, it still has the following defects: (1) There are many repetitive sequences in the detection probe prepared by using artificial chromosomes, which affects the specificity of hybridization and causes high background Fluorescence; (2) The detection steps are complicated, the probe sequence is too long, and the hybridization between the probe and the sample often takes 12-16 hours to ensure sufficient hybridization, which is time-consuming and laborious; (3) Some optimized probe sequences are too short, resulting in a decrease in detection specificity And the signal is weak and difficult to be detected

Method used

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  • BCR/ABL gene fusion and ASS gene deletion detection kit
  • BCR/ABL gene fusion and ASS gene deletion detection kit
  • BCR/ABL gene fusion and ASS gene deletion detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Kit composition

[0029] The BCR / ABL fusion gene detection kit described in this embodiment mainly includes:

[0030] 1. Fluorescent dye-labeled probe

[0031] The probes include a first set of probes for the ASS and ABL genes, a second set of probes for the upstream gene sequence of the M-BCR break site of the BCR gene, and a third set of probes for the full BCR gene sequence. The probe is obtained by using human genomic DNA as a template and using 90 pairs of specific primer pairs to perform PCR amplification reaction, and the preparation method is as follows:

[0032] 1. Design of amplification primers

[0033] In order to detect the mutations of ASS, ABL and BCR genes on chromosome 9 and 22, three sets of amplification primers were designed respectively: the first set of amplification primers for ASS and ABL genes, and the M-BCR break site for BCR gene Click the second set of amplification primers for the upstream gene sequence and the third set of amplification p...

Embodiment 2

[0058] Example 2 Using Example 1 Kit A to detect clinical samples

[0059] 1. Sample pretreatment

[0060] 1. Collect 1-1.5ml of peripheral blood from the patient with heparin sodium anticoagulation tube and centrifuge at 2000rpm for 5min;

[0061] 2. Discard the supernatant, add 5-10ml of 0.075M KCl solution, blow evenly, and treat for 30 minutes at 37°C for hypotonicity;

[0062] 3. Add 1ml of fresh fixative solution (methanol / glacial acetic acid 3:1), pipette evenly, and let stand for 5 min;

[0063] 4. Centrifuge at 2000rpm for 5min, discard the supernatant;

[0064] 5. Add 10ml of fresh fixative (methanol / glacial acetic acid 3:1), pipette evenly, and let stand for 10 minutes;

[0065] 6. Centrifuge at 2000rpm for 5min, discard the supernatant;

[0066] 7. Add 10ml of fresh fixative (methanol / glacial acetic acid 3:1), pipette evenly, centrifuge at 2000rpm for 5min, discard the supernatant;

[0067] 8. Repeat step 7 twice, and finally add 10ul fresh fixative (methanol / glacial acetic acid...

Embodiment 3

[0099] Example 3 Using Example 1 Kit B to detect clinical samples

[0100] 1. Sample pretreatment

[0101] Consistent with Example 2

[0102] Two, FISH detection

[0103] Consistent with Example 2.

[0104] Three, the result judgment standard

[0105] The criteria for determining the results of kit B of the present invention are as follows:

[0106] 1. Count 100 cells in each sample. If the number of positive cells is less than 3 (3 / 100 or <3%), the sample is judged as negative.

[0107] 2. Count 100 cells in each sample. If the number of positive cells is more than 5 (5 / 100 or >5%), the sample is judged as positive.

[0108] 3. Count 100 cells per sample. If the number of positive cells is between 3-5 (3-5%), another reader is required to count 100 cells. Summarize the number of counted cells and the number of positive cells by two readers, that is, out of a total of 200 cells, if the total number of positive cells is less than 8 (<4%), the sample is judged as negative, if the total numbe...

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Abstract

The invention discloses a BCR / ABL gene fusion and ASS gene deletion detection kit. The kit comprises a first group of probes labeled with dyes and aiming at ASS and ABL gene sequences, and a second group of probes for detecting an upstream gene sequence of an M-BCR breaking site of a BCR gene. 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 genome DNA as a template and amplification primers. The length of the FISH 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 BCR / ABL gene fusion and ASS gene deletion 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, and specifically relates to a BCR / ABL gene fusion and ASS gene deletion detection kit. Background technique [0002] Chronic Myelogenous Leukemia (CML) is a malignant clonal proliferative disease of the blood system that occurs in hematopoietic stem cells. The Philadelphia chromosome (Ph) or (and) BCR / ABL gene rearrangement can be found in the affected cell lines. The Philadelphia chromosome or (and) BCR / ABL gene rearrangement due to t(9;22)(q34;q11.2) has important diagnostic and prognostic significance in hematological tumors, and appears in more than 90% of CML, 30 % Of adult acute lymphoblastic leukemia (Acute lymphoblastic leukemia, ALL), 2%-10% of childhood ALL, and a small number of acute myelogenous leukemia (AML) and multiple myeloma (MM) patients . [0003] The BCR gene located at 22q11 and the ABL gene located at 9q34 translocate each other to form a ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6841C12Q1/6886C12Q2600/118C12Q2600/156C12Q2563/107
Inventor 朱蓉吴诗扬廖传荣
Owner SUREXAM BIO TECH
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