RNA SNP Detection Method With Improved Specificity Based on Dual-competitive-padlock-probe
10.16476/j.pibb.2024.0101
- VernacularTitle:基于双竞争挂锁探针改进RNA SNP检测特异性的方法
- Author:
Qin-Qin ZHANG
1
;
Jin-Ze LI
1
;
Wei ZHANG
1
;
Chuan-Yu LI
1
;
Zhi-Qi ZHANG
1
;
Jia YAO
1
;
Hong DU
2
;
Lian-Qun ZHOU
1
;
Zhen GUO
1
Author Information
1. School of Biomedical Engineering (Suzhou), University of Science and Technology of China, Hefei230026, China
2. Department of Clinical Laboratory, The Second Affiliated Hospital of Soochow University, Suzhou215004, China
- Publication Type:Journal Article
- Keywords:
RNA;
single nucleotide polymorphism;
genotyping;
rolling circle amplification;
dual padlock probe
- From:
Progress in Biochemistry and Biophysics
2024;51(11):3021-3033
- CountryChina
- Language:English
-
Abstract:
ObjectiveThe detection of RNA single nucleotide polymorphism (SNP) is of great importance due to their association with protein expression related to various diseases and drug responses. At present, splintR ligase-assisted methods are important approaches for RNA direct detection, but its specificity will be limited when the fidelity of ligases is not ideal. The aim of this study was to create a method to improve the specificity of splintR ligase for RNA detection. MethodsIn this study, a dual-competitive-padlock-probe (DCPLP) assay without the need for additional enzymes or reactions is proposed to improve specificity of splintR ligase ligation. To verify the method, we employed dual competitive padlock probe-mediated rolling circle amplification (DCPLP-RCA) to genotype the CYP2C9 gene. ResultsThe specificity was well improved through the competition and strand displacement of dual padlock probe, with an 83.26% reduction in nonspecific signal. By detecting synthetic RNA samples, the method demonstrated a dynamic detection range of 10 pmol/L-1 nmol/L. Furthermore, clinical samples were applied to the method to evaluate its performance, and the genotyping results were consistent with those obtained using the qPCR method. ConclusionThis study has successfully established a highly specific direct RNA SNP detection method, and provided a novel avenue for accurate identification of various types of RNAs.
