Primer, probe and kit for quantitatively detecting BCR-ABL1 fusion gene

A technology of BCR-ABL1 and BCR-BAL1, which is applied in the field of probes and kits, and primers for quantitative detection of BCR-ABL1 fusion genes, which can solve the problems of cumbersome operation, limited throughput, and high cost of digital PCR systems

Pending Publication Date: 2021-11-30
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cost of digital PCR system is high, the throughput is limited, the operation is cumbersome, and there is a lack o

Method used

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  • Primer, probe and kit for quantitatively detecting BCR-ABL1 fusion gene
  • Primer, probe and kit for quantitatively detecting BCR-ABL1 fusion gene
  • Primer, probe and kit for quantitatively detecting BCR-ABL1 fusion gene

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0113] Example 1: Kits and detection methods

[0114] This embodiment provides a composition ingredient, packaging, and quantity (20 reaction / box) of the kit of the KR-ABL1 fusion gene quantitative detection, as shown in Table 8.

[0115] Table 8. Composition ingredients, packages and quantities of the kit

[0116]

[0117]

[0118] This embodiment provides a method of quantifying the quantitative detection of BCR-ABL1 fusion gene using the above kit. The specific implementation steps are as follows:

[0119] Step 1: Sample RNA template extraction and quality detection

[0120] 1) Extraction of peripheral blood in patients with leukemia is placed in the blood collection tube placed in EDTA or citronite anticoagulant, and the mark ensures that the label information is not mistaken, 4 ° C is stored at 4 ° C.

[0121] 2) Nucleic acid extraction using a nucleic acid extraction or purification kit (Yuesui: 20170583).

[0122] 3) The concentration of the nucleic acid after the ext...

Example Embodiment

[0161] Example 2: Detection sensitivity and lowest detection rate test kit

[0162] Selection of calibrated concentrations near the detection limit 500copies / BCR-ABL1 main type BCR-ABL1 samples L1 and L2 mL sample of the minor type is verified. A nucleic acid extraction according to step, the step of detecting two pairs of samples, each test tube to make 10 copies. Operating strictly according to kit instructions, on a real-time PCR detection system. Reading results analyzed according to three pairs of the end of the amplification step, step four determination results.

[0163] Sensitivity and lowest detection limit for the detection of the present invention, test results are in Table 12.

[0164] The limit of detection results in Table 12 kit

[0165]

[0166] The detection sensitivity of the kit results with theoretical values, good detection sensitivity; can be stably detected corresponding fusion gene, and the positive rate was 100%.

Example Embodiment

[0167] The kit of reproducibility: Example 3

[0168] The copy number of the measured control samples, reference materials were prepared repetitive four, numbered respectively R1-R4. R1-R2 by mixing the main type BCR-ABL1 (P210 Keep) positive sample nucleic acid and 10ng / μL negative samples from the nucleic acid; R3-R4 mixture of secondary type BCR-ABL1 (P190) positive sample nucleic acid and 10ng / μL negative sample nucleic acid made. The main type BCR-ABL1 (P210 Keep) and secondary type BCR-ABL1 (P190) a nucleic acid samples undergo digital PCR to confirm copy number.

[0169] R1-R4 to be prepared is the subject sample, detection reproducibility reference product (R1-R4), detection is repeated 10 times, 10 times and the coefficient of variation of concentration of the sample values ​​of statistical calculations. R1-R2 are the detection result of the main type BCR-ABL1 (P210 Keep) fusion gene-positive; R3-R4 test results were minor type BCR-ABL1 (P190) fusion gene-positive; R1...

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Abstract

The invention provides a primer, a probe and a kit for quantitatively detecting a BCR-ABL1 fusion gene. Specifically, a real-time fluorescent quantitative PCR technology (RQ-PCR) is utilized to design a specific primer and a probe of a major type (P210) and a minor type (P190) of the BCR-ABL1 fusion gene, target amplification of a target gene is achieved, and rapid and specific detection of the two fusion genes is achieved. The kit contains an artificially synthesized large fragment containing a major type (P210) or minor type (P190) fusion locus of the BCR-ABL1 fusion gene as a quantitative reference substance, and the fusion gene in a patient in each treatment stage can be quantitatively detected.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a primer, a probe and a kit for quantitatively detecting the BCR-ABL1 fusion gene. Background technique [0002] Leukemia, also known as blood cancer, is a malignant clonal disease of hematopoietic stem cells. Leukemia cells are produced in the bone marrow and then spread to the blood and throughout the body. According to foreign statistics, leukemia accounts for about 3% of the total incidence of tumors, and is the most common malignant tumor in children and young people. The survey results of the incidence of leukemia in my country show that the annual incidence rate of leukemia is 2.76 / 100,000. Among all leukemias, the incidence rate of acute myeloid leukemia is the highest (1.62 / 100,000), followed by acute lymphoblastic leukemia (0.69 / 100,000). 10,000), chronic myeloid leukemia third (0.36 / 100,000). Among the subtypes of acute myeloid leukemia, the incide...

Claims

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Application Information

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IPC IPC(8): C12Q1/686C12Q1/6886C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/166C12Q2563/107C12Q2545/114C12Q2561/113
Inventor 蒋析文朱小亚王丽芳魏如涛杨美华
Owner DAAN GENE CO LTD
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