Multiplex PCR-Based Targeted Sequencing for Broad-Spectrum Pathogen Surveillance in Imported Febrile Travelers at Shenzhen Ports

Objective: To establish and evaluate a multiplex PCR-based targeted next-generation sequencing (tNGS) assay for broad-spectrum pathogen detection in imported infectious disease surveillance. Methods: A targeted sequencing panel covering 101 pathogens associated with imported infectious diseases was constructed, including viruses, bacteria, atypical bacteria, and parasites. Primer concentrations were optimized to improve amplification uniformity. Analytical performance was evaluated using dilution series, repeatability testing, specificity panels, and quantitative polymerase chain reaction (qPCR)-positive clinical reference samples. The assay was further applied to a clinical cohort of 167 samples, including 110 clinical surveillance samples and 57 qPCR-negative febrile samples. Results: After primer balancing, the optimized assay showed stable amplification uniformity, with all sequencing reactions exceeding the predefined 92% threshold. In analytical sensitivity testing, Zika virus and influenza A virus H3 subtype were both detected at 2 × 102 copies/mL; this limit of detection was determined for these two targets and was not established for the entire panel. Specificity evaluation demonstrated accurate target identification without false-positive pathogen calls. Compared with qPCR, the assay showed 96.7% concordance in 30 qPCR-positive reference samples. In the clinical cohort, 115 of 167 samples were positive by tNGS, corresponding to a detection rate of 68.9%. Among the 57 qPCR-negative febrile samples, pathogens were detected in 31 cases (54.4%), including viral–bacterial and bacterial–bacterial co-detection patterns. Cycle threshold (Ct) values showed a weak inverse correlation with log10-transformed sequencing reads (Spearman rho = −0.29, p = 0.126). Conclusions: The multiplex PCR-based tNGS assay demonstrated broad pathogen coverage and stable amplification uniformity. Analytical sensitivity was evaluated using the Zika virus and influenza A virus H3 subtype only. The assay showed high concordance with qPCR in the tested positive samples and enabled detection of mixed pathogens in qPCR-negative febrile samples. It may serve as a useful supplementary tool for pathogen screening and imported infectious disease surveillance.