Microinjection molded microwell array-based portable digital PCR system for the detection of infectious respiratory viruses

초록

In molecular diagnostics, the digital polymerase chain reaction (dPCR) has been considered a promising point-of-care testing (POCT) method for the rapid and accurate analysis of respiratory infections. To improve its practical applicability, it is necessary to develop a mass-producible and reproducible dPCR system for nucleic acid partitioning; additionally, the system must provide a customized portable analysis. In this study, we report an advanced mass-production method for the fabrication of microwell array-based dPCR chips suitable for nucleic acid partitioning and a compact fluorescence signal analysis dPCR system. Based on metal mold fabrication, different microwell sizes with diameters in the 100-200 mu m range and pitches in the 200-400 mu m range are designed and successfully fabricated using photolithography, metal electroplating, and injection molding techniques. Additionally, a battery-operated dPCR system utilizing digitalized fluorescence signal analysis is developed for on-site detection. To verify the chip and system applicability, the infectious human coronavirus is analyzed using different nucleic acid concentrations. By evaluating the performance of the dPCR chips and system, accurate and quantitative virus analysis results are obtained, verifying the portability, easy use, and reproducibility of the chips and system. Furthermore, the detection results obtained using the fabricated chips and the developed system are similar to the results obtained using commercially available systems, verifying that the proposed dPCR chips and system exhibit sensitivity, accuracy, reliability, and reproducibility in the quantitative molecular analysis of infectious diseases.

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