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Human leukemia BCR-ABL fusion mutation one-step detection kit

A detection kit and fusion mutation technology, applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of inability to achieve effective monitoring of minimal residual disease, insufficient detection sensitivity and accuracy, and accurate detection Insufficient sensitivity and sensitivity, etc., to achieve the effect of simple and efficient detection process, high accuracy and high accuracy

Inactive Publication Date: 2021-12-14
广东永诺医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ordinary PCR or direct sequencing after reverse transcription is the most direct method to detect BCR-ABL fusion gene, but the detection sensitivity is low, and it is often used in newly diagnosed patients
The detection cost of qPCR technology is low, but it relies on standard reference products in quantitative detection, and is easily affected by the efficiency of primer amplification, resulting in insufficient detection sensitivity and accuracy, and cannot effectively monitor minimal residual diseases
In summary, the detection accuracy and sensitivity of the existing BCR-ABL fusion gene or Ph-positive chromosome detection methods are not high

Method used

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  • Human leukemia BCR-ABL fusion mutation one-step detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] 1. A one-step detection kit for human leukemia BCR-ABL fusion mutation, which contains:

[0055] (1) The master mix for droplet digital PCR is a one-step ultrasensitive droplet digital PCR master mix: including buffer, dNTPs, Taq enzyme, reverse transcriptase, etc. (product of Guangdong Yongnuo Medical Technology Co., Ltd., Cat. No. S0200020401, used together with MicroDrop-100 Microdrop Digital PCR System);

[0056] (2) BCR-ABL P210 mutation detection mixture, including BCR-ABL P210 mRNA primer and probe combination, and internal standard gene GUSB mRNA primer and probe combination:

[0057] The sequence of described BCR-ABL P210 mRNA primer and probe combination is as follows:

[0058] BCR-ABL-P210 F1a: 5'-CCGCTGACCATCAAYAAG-3';

[0059] BCR-ABL-P210 R5: 5'-CCAACGAGCGGCTTCA-3';

[0060] BCR-ABL-P210 P5:5'-FAM-CCAGTAGCATCTGACTTTGAG-MGB-3';

[0061] In the mRNA primer and probe combination of BCR-ABL-P210, the working concentration of the primer is 500-1000nM, prefe...

Embodiment 2

[0094] Embodiment 2: Carry out sensitivity and the minimum mutation ratio detection limit test experiment with the kit developed by the MicroDrop-100 digital PCR platform with the plasmid sample, the specific steps are:

[0095] (1) Commercially synthesized BCR-ABL P210 positive plasmid (concentration x10 5 cp / μL) with internal standard GUSB plasmid (concentration x10 5 cp / μL) as the mother solution, prepared by gradient dilution according to the theoretical mutation ratio, and prepared 5 tubes of mixed solutions with BCR-ABL P210 / GUSB% mutation ratios of 10%, 1%, 0.1%, 0.01%, and 0.001%;

[0096] (2) Take 4 μL of each of the above 5 tubes of BCR-ABL P210 / GUSB% mutation ratio mixture as a sample, and perform detection according to the detection steps and methods of the kit;

[0097] (3) The test results are as follows: Figure 5 The one-dimensional diagram and Table 2 show:

[0098] Table 2

[0099] sample name Total Droplets Channel 1 P210 copy number Chan...

Embodiment 3

[0104] Example 3: The accuracy testing experiment of the kit developed on the MicroDrop-100 digital PCR platform was carried out with the European Bureau of Standards BCR-ABL pDNA calibrator ERM-AD623 plasmid sample

[0105] (1) The test sample is the European Bureau of Standards BCR-ABL pDNA calibrator ERM-AD623. The operation steps are tested according to the kit method. The obtained results are shown in Table 4 below:

[0106] Table 4

[0107]

[0108] (2) According to the results described in the above table, the kit of the present invention detects the international standard product, and the result shows that it is consistent with the theoretical value R2>0.99, the detection result is highly accurate, and the copy number is within the range of the theoretical value.

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a human leukemia BCR-ABL fusion mutation one-step detection kit based on digital PCR technology one-step detection. The human leukemia BCR-ABL fusion mutation one-step detection kit comprises: premixed liquid for microdroplet type digital PCR; a BCR-ABL P210 mutation detection mixed solution which comprises a BCR-ABL P210 mRNA (messenger Ribonucleic Acid) primer and probe combination and a primer and probe combination of an endogenous reference gene GUSB (Glutathione Universal Serial Bus) mRNA (messenger Ribonucleic Acid); and positive control, negative control and blank control. On a MicroDrop-100 digital PCR platform, RNA extracted from a peripheral blood or bone marrow sample is directly used as a template through a one-step method, additional experiments for reverse transcription of RNA into cDNA are not needed, an absolute quantitative result can be obtained without concentration of RNA and detection of target gene copy number in the RNA sample, a standard curve does not need to be established, the single-hole detection sensitivity can reach 0.001%, and therefore, the method has the advantages of simple and efficient detection process, absolute quantification, high specificity, high accuracy, high sensitivity and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a one-step detection kit for human leukemia BCR-ABL fusion mutation based on digital PCR technology. Background technique [0002] The Philadelphia chromosome (Ph) is a tiny chromosome produced by a balanced translocation between human chromosome 9 and chromosome 22 first discovered in 1960. This translocation leads to a parallel translocation of the long arm ABL gene (position q34) on human chromosome 9 and the long arm BCR gene (position q11) on chromosome 22, resulting in a new fusion gene BCR-ABL; BCR-ABL fusion There are three variants of the gene: P190, P210, and P230, among which P210 "also known as b3a2 / b2a2" (or "e14a2 / e13a2") is the most common type, accounting for 90% of patients with chronic myelogenous leukemia (CML ) and 1 / 3 of Ph-positive adult precursor B-cell acute lymphoblastic leukemia (precursor B-acutelymphoblastic leukemia, BALL) patients. This gene can transc...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/6886C12Q1/686C12Q2563/159
Inventor 李静芳霍云龙罗景燕许少飞赖炳权
Owner 广东永诺医疗科技有限公司
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