A Novel miRNA Detection Method Using Loop-Mediated Isothermal Amplification

Saiwei Wu; Abdu Ahmed Abdullah Al-Maskri; Qun Li; Jiatong Liu; Sheng Cai

doi:10.1155/2023/6624884

A Novel miRNA Detection Method Using Loop-Mediated Isothermal Amplification

Int J Anal Chem. 2023 Sep 12:2023:6624884. doi: 10.1155/2023/6624884. eCollection 2023.

Authors

Saiwei Wu¹, Abdu Ahmed Abdullah Al-Maskri², Qun Li¹, Jiatong Liu², Sheng Cai²

Affiliations

¹ Department of Pharmacy, The Fourth Affiliated Hospital, Zhejiang University School of Medicine, Yiwu, Zhejiang, China.
² Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.

Abstract

A novel ligation-based loop-mediated isothermal amplification has been developed for miRNA detection. Two stem-loop structure DNA linker A/B probes which hybridized with miRNA were designed to establish a rapid and ultrasensitive miRNA-LAMP system for miRNA detection. Target miR-200a was used to template the ligation of Linker A/B probes with SplintR Ligase and used as a dumbbell-shaped amplicon. By adding BIP/FIP and Bst 2.0 DNA polymerase, the LAMP reaction was carried out, which brought greatly improved amplification efficiency. The double-stranded DNA fluorescent dye EvaGreen was added for the detection of amplification product to achieve the quantification of the target miRNA. This method can detect miRNA in a linear range of seven orders of magnitude, with a detection limit of 100 fM. Therefore, this ultrasensitive miRNA-LAMP assay provides a new path for the highly sensitive quantitative analysis of miRNA, thereby bringing convenience to clinical diagnosis and prognostic research.