Increased Robustness of Single-Molecule Counting with Microfluidics, Digital Isothermal Amplification, and a Mobile Phone versus Real-Time Kinetic Measurements

Quantitative bioanalytical measurements are commonly performed in a kinetic format and are known to not be robust to perturbation that affects the kinetics itself or the measurement of kinetics. We hypothesized that the same measurements performed in a “digital” (single-molecule) format would show increased robustness to such perturbations. Here, we investigated the robustness of an amplification reaction (reverse-transcription loop-mediated amplification, RT-LAMP) in the context of fluctuations in temperature and time when this reaction is used for quantitative measurements of HIV-1 RNA molecules under limited-resource settings (LRS). The digital format that counts molecules using dRT-LAMP chemistry detected a 2-fold change in concentration of HIV-1 RNA despite a 6 °C temperature variation (p-value = 6.7 × 10–7), whereas the traditional kinetic (real-time) format did not (p-value = 0.25). Digital analysis was also robust to a 20 min change in reaction time, to poor imaging conditions obtained with a cons...