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Drop-off ddPCR method and kit for quantitatively detecting NPM1 gene mutation

A quantitative detection and kit technology, which is used in DNA/RNA fragmentation, microbial determination/inspection, biochemical equipment and methods, etc., and can solve problems such as low sensitivity, single mutation type, and inability to perform absolute quantification by real-time fluorescent quantitative PCR. , to achieve high sensitivity and specificity

Active Publication Date: 2021-08-13
ZHENJIANG NO 1 PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Sanger sequencing is considered the gold standard for detecting gene mutations, but its sensitivity is low and it is not suitable for clinical MRD detection
Next-generation sequencing has high throughput and high sensitivity, but its high cost and difficult operation make it difficult to apply single-gene MRD monitoring in clinical practice
Real-time fluorescent quantitative PCR cannot perform absolute quantification and is not suitable for MRD monitoring
The traditional ddPCR method has high sensitivity and can directly reflect the NPM1 mutation load level, but the detection of mutation types is relatively single, and only known mutations can be detected. The detection of multiple mutation types on mutation hotspots is cumbersome and easy to miss

Method used

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  • Drop-off ddPCR method and kit for quantitatively detecting NPM1 gene mutation
  • Drop-off ddPCR method and kit for quantitatively detecting NPM1 gene mutation
  • Drop-off ddPCR method and kit for quantitatively detecting NPM1 gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Taking the bone marrow samples of patients with NPM1 gene type A mutation as an example, the detection method is described in detail.

[0046] 1. Isolation of bone marrow mononuclear cells and extraction of DNA

[0047] (1) Add 5ml red blood cell lysate to the bone marrow sample, mix well, and let stand for 1min;

[0048] (2) Centrifuge at 12000×rpm for 20sec, discard the supernatant and keep the precipitate;

[0049] (3) Add 1ml Trizol and mix well by pipetting;

[0050](4) Add 200 μl of chloroform, vortex for 20 sec, let stand for 10 min, centrifuge at 12000×rpm for 1 min, and discard the supernatant;

[0051] (5) After centrifuging again at 12000×rpm, discard the supernatant and keep the precipitate, and the white precipitate of DNA at the bottom of the tube can be seen;

[0052] (6) Add 1ml of trisodium citrate to the tube, flick or invert to mix and rinse the precipitate, let stand for 20-30min, centrifuge at 12000×rpm for 5min, and discard the supernatant;

[...

Embodiment 2

[0074] Taking the bone marrow sample of a patient with a D-type mutation of the NPM1 gene as an example, the specific detection process is the same as that in Example 1. After data analysis, it can be obtained as figure 2 The experimental results and related data. In 250ng sample DNA, the concentration of FAM is 1660.10copies / μl, the concentration of VIC is 1921.90copies / μl, the calculated concentration of NPM1 D-type mutation template is 261.80copies / μl, and the allele frequency of NPM1 is 13.62%.

Embodiment 3

[0076] Taking the bone marrow sample of a patient with negative NPM1 gene mutation as an example, the specific detection process is the same as that in Example 1. After data analysis, it can be obtained as image 3 The experimental results and related data. In 250ng sample DNA, the concentration of FAM was 2078.37copies / μl, the concentration of VIC was 2078.37copies / μl, and the calculated concentration of NPM1 mutation template was 0copies / μl, which was lower than the detection limit (22.78copies / μl), so the patient’s NPM1 gene mutation was negative.

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Abstract

The invention relates to a detection method and a kit for detecting NPM1 gene mutation based on drop-offddPCR; primers and probes thereof are designed according to an NPM1 gene DNA sequence; two wild probes are designed aiming at a 12th exon mutation hot spot of an NPM1 gene; one wild probe is located on the mutation hot spot; the other wild probe is located outside the mutation hot spot; when mutation such as insertion, replacement and deletion exists in the mutation hot spot, the wild probe located at the mutation hot spot cannot be tightly combined with a template, so that the kit can detect various mutations of the mutation hot spot of the 12th exon of the NPM1 gene only by using two pairs of primer probes; and the kit is high in sensitivity and can be used for MRD monitoring.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and kit for quantitatively detecting NPM1 gene mutation. Background technique [0002] Nucleophosmin 1 (nucleophosmin, NPM1) gene is located on human chromosome 5q35, with 12 exons in total, and its protein consists of 294 nucleotides. NPM1 mutation is one of the most common genetic mutations in acute myeloid leukemia (AML). NPM1 mutations are detected in 25%–35% of AML cases, over 50% in cytogenetically normal AML (CN-AML), and in myeloproliferative and myeloid dysplastic disorders There are also discoveries. So far, about 50 NPM1 exon 12 mutations have been found, most of which are insertions between nucleotides 863 and 864. NPM1 mutations are very stable during the disease process and thus are one of the important molecular markers for better diagnosis and disease monitoring in AML. [0003] Minimal residual disease (MRD) refers to submicroscopic lesions that still ex...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/6886C12Q1/686C12Q2600/156C12Q2545/114C12Q2563/159
Inventor 金晔钱军林江徐子浚闻向梅马吉春
Owner ZHENJIANG NO 1 PEOPLES HOSPITAL
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