SMART PCR for adaptive low-abundance oral bacterial DNA detection through real-time fluorescence-guided thermal regulation

Quantitative polymerase chain reaction (qPCR) is widely used for oral pathogen detection and molecular diagnostics, where reliable identification of low-abundance bacterial DNA remains challenging, yet its thermal-cycling protocol is typically predefined before the reaction begins and then repeats unchanged. Such static, open-loop control does not match the continuously evolving reaction state and can limit amplification efficiency within each cycle, particularly for low-abundance targets. Here, we report SMART (Self-Monitoring and Real-Time optimization) PCR, a closed-loop qPCR biosensing-actuation strategy that regulates the annealing/extension step during amplification. The strategy integrates a continuous fluorescence monitoring (CFM) system with a causal temporal-convolutional decision model that classifies within-cycle fluorescence dynamics during the annealing/extension step. The model converts real-time fluorescence trajectories into control signals that determine whether annealing/extension should be maintained or whether the reaction should proceed to the next cycle. In a tested two-step qPCR assay using a synthetic oral bacterial DNA target, annealing/extension duration is identified as the dominant controllable parameter affecting threshold cycle ( C T ) values. The SMART PCR protocol adaptively extends annealing/extension when the real-time trajectory indicates that additional reaction time is useful, rather than applying a uniformly prolonged step across all cycles. Compared with a conventional fixed protocol, SMART PCR produces earlier threshold crossing and improved analytical sensitivity under the present assay conditions, with the largest C T improvement reaching 3.50 cycles at the lowest tested synthetic-template concentration, from 10 fM with fixed qPCR to 1 fM with SMART PCR. These results demonstrate a practical fluorescence-sensing, decision-making and thermal-actuation framework for state-responsive nucleic-acid amplification. These results support SMART PCR as a promising adaptive amplification framework for improving low-template analytical amplification performance, while further validation using clinical samples is required before diagnostic application.