Citation: QI Tong, SONG Chang, CHEN Wen-Hui, TANG Liang-Xiu, JU Jia-He, SHEN Wei, KONG De-Zhao, TANG Sheng. Duplex Specific Nuclease-Enabled Isothermal Signal Amplification Combined with High-Performance Liquid Chromatography for Highly Sensitive Detection of Dual-Target MicroRNAs as Disease Biomarkers[J]. Chinese Journal of Analytical Chemistry, ;2021, 49(2): 216-225. doi: 10.19756/j.issn.0253-3820.201298 shu

Duplex Specific Nuclease-Enabled Isothermal Signal Amplification Combined with High-Performance Liquid Chromatography for Highly Sensitive Detection of Dual-Target MicroRNAs as Disease Biomarkers

1.
School of Environmental and Chemical Engineering, Jiangsu University of Science and Technology, Zhenjiang 212003, China;
2.
School of Grain Science and Technology, Jiangsu University of Science and Technology, Zhenjiang 212003, China

Corresponding author: TANG Sheng, tangsheng.nju@gmail.com
Received Date: 24 May 2020
Revised Date: 13 November 2020

Fund Project: Supported by the National Natural Science Foundation of China (Nos.21605105, 21705060), the Natural Science Foundation of Jiangsu Province, China (No.BK20170570) and the Six Talent Peaks Project in Jiangsu Province, China (No.SWYY-021).

A method for selective separation and highly sensitive quantitation of dual-target miRNAs as disease biomarkers by duplex specific nuclease (DSN)-enabled signal amplification strategy combined with high-performance liquid chromatography (HPLC) was developed. The enzyme-based isothermal amplification strategy enhanced the signals of the target miRNAs and solved the problem of low sensitivity during the detection of nucleic acids utilizing conventional HPLC. To obtain an effective separation of the signals of different miRNAs, DNA probes which were immobilized on magnetic beads were designed with different lengths and base sequences. Effective magnetic separation minimized the background noise and expanded the dynamic range. Consequently, the assay achieved a detection limit of 0.33 fmol/L for miRNA-155 and 0.24 fmol/L for miRNA-21, respectively. The proposed assay was successfully applied to detection of miRNA-155 and miRNA-21 in serum samples from lupus erythematosus, cervical cancer and ovarian cancer patients. The results obtained here were comparable to those obtained by quantitative real-time polymerase chain reaction.
- MicroRNA,
- Dual-target assay,
- Fluorescence,
- Duplex specific nuclease,
- Enzyme-based isothermal amplification,
- High-performance liquid chromatography
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沈阳化工大学材料科学与工程学院沈阳 110142