Three-Dimensional Fluorescence Fingerprint Integrated PCR Enables Accurate Analysis of 9 Pathogens

Rapid and accurate identification of respiratory tract infections is a critical clinical need, with current diagnostic approaches predominantly relying on culture isolation, immunological testing, and molecular biology techniques. Although PCR-based methods have gained clinical acceptance due to their specificity and sensitivity, they are limited by the signal acquisition capabilities. Therefore, breaking through the limitations of signal acquisition capability and developing a high-throughput pathogen identification method remains an urgent challenge. In this study, we propose 3D-FLIP9 (3D Fluorescent Fingerprint Integrated PCR for Simultaneous Detection of 9 Pathogens), a high-throughput platform that combines 3D fluorescent fingerprinting and PCR. This method enables the detection of 9 respiratory pathogens in a single tube with significant efficiency and convenience. 3D-FLIP9 has made a breakthrough in signal acquisition and processing, allowing for accurate pathogen identification based on distinctive data points, with each pathogen presenting a unique fingerprint. The fluorescence signal is detectable at the target identification position even at 10 copies/μL. Moreover, 3D-FLIP9 has demonstrated substantial effectiveness in a total of 136 clinical samples (overall AUC = 97%). This work underscores the critical role of 3D-FLIP9 in the simultaneous detection of multiple respiratory pathogens, offering significant application value for the prevention and control of respiratory infections.