Development of a Multiplex Droplet Digital PCR Assay for Detection of Tick-Borne Pathogens

Tick-borne pathogens pose a significant threat to human health. In this study, a multiple droplet digital PCR (ddPCR) assay was developed to detect four tick-borne pathogens: <i>Borrelia burgdorferi sensu lato</i> (<i>Bbsl</i>), <i>Coxiella burnetii</i> (<i>C. burnetii</i>), spotted fever group Rickettsia (SFGR), and <i>Borrelia miyamotoi</i> (<i>B. miyamotoi</i>). Based on the singleplex ddPCR reaction system of <i>Bbsl</i>, the primer probes of the other three species were incorporated to develop a multiplex ddPCR reaction system. The annealing temperature and the final concentration of the primer probes were then optimized for multiplex ddPCR. The multiplex ddPCR assay was assessed for its sensitivity, specificity, repeatability, and ability to detect simulated and actual samples. The developed multiplex ddPCR approach enables the simultaneous detection of <i>Bbsl</i>, <i>C. burnetii</i>, SFGR, and <i>B. miyamotoi</i>. The positive target microtitre clusters are closely grouped and distinctly separated from each other, with the multiplex ddPCR assay demonstrating a dynamic range of five orders of magnitude. The limits of detection (LOD) for the multiplex ddPCR assay were 4 copies/20 µL for <i>Bbsl</i>, 3 copies/20 µL for <i>C. burnetii</i>, 3 copies/20 µL for SFGR, and 2 copies/20 µL for <i>B. miyamotoi</i>. The assay demonstrated high specificity, with no observed cross-reactivity against non-target pathogens. Performance was validated using both spiked samples and field-collected clinical specimens. In the evaluation of 30 ticks and 30 serum samples, the ddPCR method (in both singleplex and multiplex formats) achieved higher positive detection rates for all four target pathogens compared to quantitative real-time PCR (qPCR). In addition, the detection proportions of multiplex and singleplex ddPCR were consistent. Multiplex ddPCR can detect low DNA concentrations in samples and enables the absolute quantification of <i>Bbsl</i>, <i>C. burnetii</i>, SFGR, and <i>B. miyamotoi</i>, providing a novel detection approach for the clinical diagnosis of tick-borne diseases.