Abstract
We describe the development, optimization, and validation of a multiplex droplet digital PCR (ddPCR) assay for the simultaneous detection of Babesia, Bartonella, and Borrelia spp. DNA from several sample matrices, including clinical blood samples from animals and humans, vectors, in-vitro infected human and animal cell lines, and tissues obtained from animal models (infected with Bartonella and/or B. burgdorferi). The multiplex ddPCR assay was able to detect 31 Bartonella, 13 Borrelia, and 24 Babesia species, including Theileria equi, T. cervi, and Cytauxzoon felis. No amplification of Treponema or Leptospira spp. was observed. Sensitivity of 0.2–5 genome equivalent DNA copies per microliter was achieved for different members of the Bartonella and Borrelia genus, depending on the species or matrix type (water or spiked blood DNA) tested. The ddPCR assay facilitated the simultaneous detection of co-infections with two and three vector-borne pathogens comprising four different genera (Babesia, Bartonella, Borrelia, and Theileria) from clinical and other sample sources.
Highlights
Droplet digital polymerase chain reaction (PCR), a relatively new molecular assay, was originally developed for research and diagnostic applications in the areas of cancer and gene expression [1,2,3,4,5,6,7,8]
We describe the development of a multiplex droplet digital PCR assay for the simultaneous detection of Babesia, Bartonella, and Borrelia species (BBB Droplet digital PCR (ddPCR)) using the Bio-Rad QX One Droplet Digital PCR system
DNA from previously characterized or as yet to be characterized Babesia (Piroplasma), Bartonella, and Borrelia spp., including positive and negative research and diagnostic samples from various host animals and humans submitted to the Vector-Borne Diseases Diagnostic Laboratory (VBDDL) and the Intracellular Pathogens Research Laboratory (IPRL), both at the College of Veterinary Medicine, North Carolina State University, were used to test the specificity of this BBB ddPCR assay
Summary
Droplet digital PCR (ddPCR), a relatively new molecular assay, was originally developed for research and diagnostic applications in the areas of cancer and gene expression [1,2,3,4,5,6,7,8]. Serology and PCR-based assays (real-time and conventional PCR) have been used diagnostically, contributing to increased recognition of babesiosis as a more prevalent and significant tick-borne infection in animals and humans worldwide [37,40,41,42,43,44,45]. Pathogens 2021, 10, 1462 fastidious nature, complex growth requirements, cyclical, relapsing low bacteremia, and their ability to invade several cells types to subvert/evade the immune system (often leading to long delays in seroconversion and negative serology test results) [87,88,89,90,91,92,93,94,95,96,97,98], specialized diagnostic modalities, including a recently described Bartonella droplet digital PCR detection assay, are critically needed to improve diagnostic sensitivity [17,18,99]. Several sample matrices were tested, including experimentally infected human and animal cell lines, spiked blood samples, animal (both domestic and wildlife) and human pre-characterized clinical samples (blood and tissue), and naturally infected sand-fly and tick species
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