Development and Application of a Multiplex qPCR Rapid Detection System for Syndromic Detection of Tick-Borne Pathogen Coinfections

Polymicrobial coinfections are increasingly becoming prevalent within Ixodidae tick vectors, which considerably complicates the global burden of tick-borne diseases. This reality necessitates rapid and accurate detection methodologies capable of concurrent pathogen detection. We engineered a multiplex quantitative polymerase chain reaction (qPCR) assay targeting Spotted Fever Group Rickettsiae (SFGR), Babesia microti, severe fever with thrombocytopenia syndrome virus, and Anaplasma phagocytophilum. Performance was evaluated on plasmid standard samples and 2050 field-collected ticks, benchmarking against established conventional PCR assay. The assay achieved 10 copies sensitivity, confirmed by probit regression (limit of detection95: 12.4–15 copies/µL) and robust quantification (R2 = 0.975–0.988, E = 95.630–101.668%). Receiver operating characteristic analysis suggested excellent preliminary accuracy (area under the curve: 1.000; sensitivity: 100.00%; specificity: 98.57–100.00% at a pre-specified Ct cutoff of 35). Clinical validation showed high concordance with conventional PCR for single pathogens (κ = 0.977–1.000; p < 0.001) and 100% agreement in quadruplex coinfection detection within the limited positive cases. Therefore, multiplex qPCR technology emerges as a sensitive, rapid, extremely specific, and efficient method for real-time syndromic surveillance of tick-borne polymicrobial infections, which enhances detection in regions burdened by overlapping vectors and interconnected zoonoses.