DNA-Aptamer-Based qPCR Using Light-Up Dyes for the Detection of Nucleic Acids

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 Ct vs log₁₀ (DNA amount) standard curves, which have a linearfit with R² value > 0.99. This qPCR technique was validated by quantifying gene targets from Streptococcus zooepidemicus (SzhasB) and Mycobacterium tuberculosis (MtrpoB). 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.