Sophisticated and precise: design and implementation of a real-time optical detection system for ultra-fast PCR

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) has become indispensable in the realm of disease nucleic acid screening and diagnostics, owing to its remarkable precision and sensitivity, in which the real-time fluorescence detection system plays an extremely critical role. To solve the problems of long time and slow speed of traditional nucleic acid detection, PCR systems are evolving towards ultra-rapid configurations. Nonetheless, most extant ultra-rapid PCR systems either depend on endpoint detection for qualitative assessments due to inherent structural or heating constraints or circumvent the challenge of adapting optical systems to expeditious amplification systems, resulting in potential shortcomings in assay efficacy, volume, or expense. Consequently, this study proposed a design of a real-time fluorescence detection system for ultra-fast PCR, capable of executing six channels of real-time fluorescence detection. Through the meticulous calculation of the optical pathway within the optical detection module, effective regulation of system dimensions and the cost was accomplished. By devising an optical adaptation module, the signal-to-noise ratio was enhanced by approximately 307% without compromising the PCR temperature alteration rate. Ultimately, by employing a fluorescence model that accounted for the spatial attenuation effect of excitation light, as proposed herein, fluorescent dyes were arranged to evaluate the repeatability, channel interference, gradient linearity, and limit of detection of the system, which proved that the system had good optical detection performance. Finally, the real-time fluorescence detection of human cytomegalovirus (CMV) under 9 min ultra-fast amplification was achieved by a complete ultra-fast amplification experiment, which further validated the potential of the system to be applied to rapid clinical nucleic acid detection.