Kit for detecting gene mutations in acute lymphoblastic leukemia based on fluorescence quantitative PCR and method

An acute lymphocyte and fluorescent quantitative technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of inability to detect large fragments of genome copy number variation, and avoid false positives, Easy operation and high sensitivity effect

Pending Publication Date: 2020-08-14
南京实践医学检验有限公司
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  • Application Information

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Problems solved by technology

Because the amplification of the first-generation sequencing technology is limited to a fragment length of about 1kb, it cannot detect the copy number variation of large fragments of the genome, so false negatives will occur

Method used

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  • Kit for detecting gene mutations in acute lymphoblastic leukemia based on fluorescence quantitative PCR and method
  • Kit for detecting gene mutations in acute lymphoblastic leukemia based on fluorescence quantitative PCR and method
  • Kit for detecting gene mutations in acute lymphoblastic leukemia based on fluorescence quantitative PCR and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] Prepare PAX5 gene (1-10) and IKZF1 gene (2-8) mutation detection kits, specifically comprising the following steps:

[0135] 1. Synthetic primers and probe sequences

[0136] Synthesize PCR primer probe sequences SEQ ID NO.1-SEQ ID NO.51, wherein the 5' end of the specific probe sequence is labeled with a FAM fluorescent group. Synthesize the internal standard primer probe sequence SEQ ID NO.52-SEQ ID NO.54, wherein, the internal standard probe sequence SEQ ID NO.54 is labeled with a FAM fluorescent group at the 5' end and a BHQ1 quencher at the 3' end group.

[0137] The above primer sequences (or internal standard primers) were respectively prepared into 100 pmol / ul stock solution for storage, and the above probe sequences (internal standard probes) were prepared respectively for storage in stock solution.

[0138] 2. Preparation of fluorescent quantitative PCR reaction system

[0139] Prepare mutation detection reaction systems containing No. 1-17 reagents respect...

Embodiment 2

[0144] Use the PAX5 and IKZF1 gene mutation detection kit prepared in Example 1 to detect the samples to be tested.

[0145] In this embodiment, 16 cases (numbering B1-B16)) of newly diagnosed, refractory and relapsed B-ALL patients (16 patients including children and adults) were collected and tested through informed consent (children were relatives). Agree to detect) blood samples and extract genomic DNA therefrom, detect whether there is corresponding gene mutation in the sample to be tested with the PAX5 and IKZF1 gene mutation detection kit that obtains in embodiment 1, concrete operation steps are:

[0146] 1. Extraction of Genomic DNA from Samples

[0147] Using a DNA extraction kit (The Blood DNA Kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) was used to extract the genomic DNA of the blood samples of the above-mentioned B-ALL patients and the genomic DNA of normal persons. NANO300 was used to detect the concentration of DNA. The A260 / A280 value of ...

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Abstract

The invention discloses a kit for detecting gene mutations in acute lymphoblastic leukemia based on fluorescence quantitative PCR. The kit comprises a PCR primer pair, a probe, a 2x PCR Nucleotide Mixreaction solution and RNase-Free ddH20. The kit has the advantages that a fluorescence quantitative PCR method is adopted to carry out specific amplification detection on PAX5 and IKZF1 gene mutationregion sequences, while high sensitivity and specificity are ensured, more mutation sites are considered. The detection kit provided by the embodiment is high in sensitivity, good in specificity andsimple to operate, and is a human PAX5 and IKZF1 gene mutation detection kit with excellent performance.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a kit and method for detecting gene mutations in acute lymphoblastic leukemia based on fluorescence quantitative PCR. Background technique [0002] Leukemia is a group of life-threatening blood and bone marrow malignancies. Acute leukemia is most prevalent in adolescents and young adults, and chronic myeloid leukemia is less common. Its diagnosis, treatment and prognosis require a comprehensive analysis of MICM from morphology, immunology, cytogenetics and molecular biology. From the perspective of molecular biology, the main detection is gene expression, fusion gene and gene variation. Among gene variation, point mutation, The variation of copy number (amplification / deletion) is the main one, and the abnormal copy number of gene PAX5 and IKZF1 is the most common variation in B-ALL. [0003] Acute lymphoblastic leukemia (ALL) is now more than 80%, but current treatment h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11C12Q1/6851
CPCC12Q1/6886C12Q1/6851C12Q2600/156C12Q2563/107C12Q2531/113C12Q2565/125
Inventor 唐春花张鹏邢宽何志健谢珍夏统前袁鸣何贵伦安雪茹李平
Owner 南京实践医学检验有限公司
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