School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing, 100083, China.
School of Chemistry and Chemical Engineering, Shaanxi Normal University, 620 Xi Chang'an Street, Xi'an, 710119, Shaanxi Province, China.
Talanta. 2020 May 15;212:120754. doi: 10.1016/j.talanta.2020.120754. Epub 2020 Jan 15.
Robust, reliable, and sensitively quantitative detection of genetic biomarkers at single-base resolution has the potential to revolutionize medical diagnostics, especially for precision medicine. Here, taking the advantages of the high specificity of ligase reaction and the powerful amplification features of the isothermally exponential amplification, we have demonstrated a novel methodology to sensitively quantify genetic biomarkers at one-base resolution. The methodology is based on the ligase reaction of two stem-loop DNA probes templated by the nucleic acid targets to form a double stem-loop DNA, which subsequently initiates the isothermally exponential amplification reaction with high amplification efficiency. With the proposed method, high sensitivity to determine as low as 0.01 fM DNA or 0.1 fM RNA targets and high specificity to detect single-base changes can be achieved. The new methodology is robust to be performed by using a pair of universal primers under isothermal conditions, which should be employed to quantitatively detect any genetic biomarkers because all DNA/RNA targets can be directly used as the templates to ligate the stem-loop DNA probes with single-base resolution.
在单碱基分辨率下稳健、可靠且灵敏地检测遗传生物标志物有可能彻底改变医学诊断，特别是在精准医疗领域。在这里，我们利用连接酶反应的高特异性和等温指数扩增的强大扩增特性，展示了一种新的方法，可以在单碱基分辨率下灵敏地定量遗传生物标志物。该方法基于两个茎环 DNA 探针与核酸靶标模板的连接酶反应，形成双链茎环 DNA，随后以高扩增效率启动等温指数扩增反应。通过该方法，可以实现对低至 0.01 fM DNA 或 0.1 fM RNA 靶标的高灵敏度检测，以及对单碱基变化的高特异性检测。该新方法在等温条件下使用一对通用引物进行时稳健可靠，由于所有 DNA/RNA 靶标都可以直接用作模板与茎环 DNA 探针进行连接，因此该方法应该被用于定量检测任何遗传生物标志物。
