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A drop-off ddPCR method and kit for quantitative detection of u2af1 gene mutation

A quantitative detection and kit technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of low sensitivity, single mutation type, difficult operation, etc., and achieve high sensitivity and strong specificity

Active Publication Date: 2022-02-22
ZHENJIANG NO 1 PEOPLES HOSPITAL
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  • 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 (Droplet Digital PCR) method has high sensitivity and can directly reflect the mutation load level of U2AF1, but the type of mutation detected is relatively single, and only known mutations can be detected. Mutation type detection is cumbersome and easy to miss

Method used

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  • A drop-off ddPCR method and kit for quantitative detection of u2af1 gene mutation
  • A drop-off ddPCR method and kit for quantitative detection of u2af1 gene mutation
  • A drop-off ddPCR method and kit for quantitative detection of u2af1 gene mutation

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Experimental program
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Effect test

Embodiment 1

[0054] Taking the bone marrow samples of patients with U2AF1 gene Q157P mutation as an example, the detection method is described in detail.

[0055] 1. Isolation of bone marrow mononuclear cells and extraction of DNA

[0056] (1) Add 5ml of erythrocyte lysate to the bone marrow sample, mix well, and let stand for 1min;

[0057] (2) Centrifuge at 12000×rpm for 20sec, discard the supernatant and keep the precipitate;

[0058] (3) Add 1ml Trizol and mix well by pipetting;

[0059] (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;

[0060] (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;

[0061] (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

[0084] Take the bone marrow samples of patients with U2AF1 gene Q157R mutation as an example. The specific detection process is the same as that in Embodiment 1, and will not be repeated here. After data analysis, it can be obtained as figure 2 The experimental results and related data.

[0085] In 250ng sample DNA, the concentration of FAM is 4712.85 copies / microliter, the concentration of VIC is 4857.83 copies / microliter, the calculated U2AF1 Q157 mutation template concentration is 144.98 copies / microliter, and the U2AF1 allele frequency is 2.98%.

Embodiment 3

[0087] Take the bone marrow samples of U2AF1 gene Q157 mutation-negative patients as an example. The specific detection process is the same as that in Embodiment 1, and will not be repeated here. After data analysis, it can be obtained as image 3 The experimental results and related data.

[0088] In 250ng sample DNA, the concentration of FAM is 4611.04 copies / microliter, the concentration of VIC is 4612.77 copies / microliter, the calculated concentration of U2AF1 Q157 mutation template is 1.73 copies / microliter, which is lower than the detection limit (8.00 copies / microliter), so The patient was negative for U2AF1 gene mutation.

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Abstract

The invention relates to a detection method and kit for detecting U2AF1 gene mutation based on drop-offddPCR. The primers and probes are designed according to the U2AF1 gene DNA sequence, and two wild One is located on the mutation hotspot, and the other is located outside the mutation hotspot. When there are insertions, substitutions, deletions and other mutations in the mutation hotspot, the wild-type probe located at the mutation hotspot cannot tightly bind to the template, so the kit is only used for Two pairs of primer probes can detect multiple mutations in the two mutation hotspots of U2AF1 gene Q157 and S34, and its sensitivity is high, which 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 U2AF1 gene mutation. Background technique [0002] U2AF1 is a cofactor of U2 ribonucleoprotein, located on the long arm of chromosome 21 (21q22.3), and belongs to a kind of RNA splicing complex protein, which contains two functional domains, namely RNA binding domain and RS structure domain, which plays a role in the splicing of pre-mRNA into functional mRNA. U2AF1 gene has a high mutation rate in myelodysplastic syndrome (MDS) patients, and 8.7% to 11.6% of patients with first-onset MDS have U2AF1 gene mutation. Studies have shown that U2AF1 gene mutations are associated with the prognosis of MDS. MDS patients with U2AF1 gene mutations have a high risk of transformation to acute myeloid leukemia (AML), and the overall survival (OS) is shorter, which usually indicates a poor prognosis. U2AF1 and other RNA splicing body encoding genes ar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6851
CPCC12Q1/6886C12Q1/6851C12Q2600/156C12Q2600/118
Inventor 金晔钱军林江徐子浚马吉春闻向梅
Owner ZHENJIANG NO 1 PEOPLES HOSPITAL
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