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

## TL;DR

A new multiplex ddPCR test was developed to detect four tick-borne pathogens simultaneously, offering higher sensitivity and accuracy than traditional methods.

## Contribution

A novel multiplex ddPCR assay for simultaneous detection of four tick-borne pathogens with high sensitivity and specificity.

## Key findings

- The assay detected all four pathogens with limits of detection as low as 2 copies/20 µL.
- Multiplex ddPCR showed higher detection rates than qPCR in clinical and tick samples.
- The method enables absolute quantification and has no cross-reactivity with non-target pathogens.

## Abstract

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: Borrelia burgdorferi sensu lato (Bbsl), Coxiella burnetii (C. burnetii), spotted fever group Rickettsia (SFGR), and Borrelia miyamotoi (B. miyamotoi). Based on the singleplex ddPCR reaction system of Bbsl, 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 Bbsl, C. burnetii, SFGR, and B. miyamotoi. 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 Bbsl, 3 copies/20 µL for C. burnetii, 3 copies/20 µL for SFGR, and 2 copies/20 µL for B. miyamotoi. 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 Bbsl, C. burnetii, SFGR, and B. miyamotoi, providing a novel detection approach for the clinical diagnosis of tick-borne diseases.