Drop-off ddPCR method and kit for quantitatively detecting U2AF1 gene mutation

Abstract

The invention relates to a detection method and a kit for detecting U2AF1 gene mutation based on drop-ffdPCR. Primers and probes of the detection method are designed according to a U2AF1 gene DNA sequence, two wild probes are designed for two mutation hotspots Q157 and S34 of a U2AF1 gene respectively, one wild probe is located on the mutation hotspot, the other wild probe is located outside the mutation hotspot, and when mutation such as insertion, replacement and deletion exists in the mutation hotspot, the wild-type probe located at the mutation hotspot cannot be tightly combined with the template, so that the kit can detect multiple mutations of the two mutation hot spots Q157 and S34 of the U2AF1 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 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...

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

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