A-406 Integration of a Rapid Real-time PCR Module into Point of Care Instruments

Abstract

Background Polymerase chain reaction (PCR) and real-time PCR (qPCR) are widely regarded as gold-standard methods for nucleic acid amplification in diagnostic applications. Despite advances in PCR instrumentation, conventional systems can require more than 30 minutes to complete an assay, limiting their utility for point-of-care testing. Microfluidic PCR platforms offer the potential for faster amplification due to reduced thermal mass and improved heat transfer, but achieving ultra-rapid thermal cycling while maintaining sensitivity and real-time fluorescence detection remains a technical challenge. This work addresses this gap by developing and evaluating an ultra-rapid real-time microfluidic PCR module capable of amplifying DNA and RNA with dramatically reduced time to result, and suitable for incorporation into point-of-care or benchtop instruments. Methods A custom RT-PCR module was developed that integrates injection-molded polycarbonate microfluidic chips with a novel thermal cycling system. The microfluidic chip incorporates a small-volume amplification chamber sealed with thin polymeric films to promote efficient heat transfer. Two thermoelectric Peltier elements clamp the amplification chamber, enabling active heating and cooling, while a strategically positioned fluorescence detector allows real-time signal acquisition during continuous thermal cycling. Thermal cycling parameters, including ramp rates and dwell times, were systematically optimized. The module was previously integrated into a point-of-care instrument and independently tested with standard, unmodified commercial RT-PCR chemistries, and then integrated into a second instrument design, demonstrating its modularity and flexibility. Results In benchtop testing, the RT-PCT module achieved heating and cooling rates of up to 12°C/s and 10°C/s, respectively, with accurate and reproducible temperature control inside the amplification chamber. Forty cycles of real-time PCR were completed in as little as 8 minutes, representing an 84% reduction in assay time compared with a standard ∼50-minute protocol. Once integrated into the first instrument, an optimized protocol completed amplification in under 15 minutes with minimal loss of efficiency. PCR efficiency for DNA amplification was measured at 99.5%, and sensitivity was comparable to that of a commercial instrument, detecting DNA concentrations down to 1pg/µL. Successful amplification was demonstrated for both forensic DNA targets and clinical SARS CoV 2 RNA. Similar performance was then demonstrated in a completely different instrument design and assay chemistry. Conclusion This study demonstrates an ultra-rapid real-time microfluidic PCR module that enables fast, reliable amplification of DNA and RNA without modifying standard assay chemistries. By combining efficient microfluidic chip design, dual Peltier-based thermal cycling, and simultaneous fluorescence detection, the module achieves substantial reductions in time to result while maintaining sensitivity and efficiency comparable to conventional laboratory instruments. The speed and compact design of the module make it well suited for integrating into point-of-care and benchtop instruments, addressing a critical demand for rapid nucleic acid testing in clinical diagnostic settings.