Goyal Shivansh, Singh Prashant, Sengupta Sudeshna, Muthukrishnan Anantha Barathi, Jayaraman Guhan
Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, Tamil Nadu 600036, India.
ACS Omega. 2023 Nov 28;8(49):47277-47282. doi: 10.1021/acsomega.3c07599. eCollection 2023 Dec 12.
Quantitative polymerase chain reaction (qPCR) is widely used in detection of nucleic acids, but existing methods either lack sequence-specific detection or are costly because they use chemically modified DNA probes. In this work, we apply a DNA aptamer and light-up dye-based chemistry for qPCR for nucleic acid quantification. In contrast to the conventional qPCR, in our method, we observe an exponential decrease in fluorescence upon DNA amplification. The qPCR method we developed produced consistent vs log (DNA amount) standard curves, which have a linearfit with value > 0.99. This qPCR technique was validated by quantifying gene targets from () and (). We show that our strategy is able to successfully detect DNA at as low as 800 copies/μL. To the best of our knowledge, this is the first study demonstrating the application of light-up dyes and DNA aptamers in qPCR.
定量聚合酶链反应（qPCR）广泛应用于核酸检测，但现有方法要么缺乏序列特异性检测，要么因使用化学修饰的DNA探针而成本高昂。在本研究中，我们将基于DNA适配体和发光染料的化学方法应用于qPCR进行核酸定量。与传统qPCR不同，在我们的方法中，DNA扩增时荧光呈指数下降。我们开发的qPCR方法产生了一致的 vs log（DNA量）标准曲线，其线性拟合值>0.99。通过对来自（）和（）的基因靶点进行定量，验证了这种qPCR技术。我们表明，我们的策略能够成功检测低至800拷贝/μL的DNA。据我们所知，这是第一项证明发光染料和DNA适配体在qPCR中应用的研究。
