Abstract
BackgroundKnowledge on the capacity of Australian ticks to carry Borrelia species is currently limited or missing. To evaluate the potential of ticks to carry bacterial pathogens and their DNA, it is imperative to have a robust workflow that maximises recovery of bacterial DNA within ticks in order to enable accurate identification. By exploiting the bilateral anatomical symmetry of ticks, we were able to directly compare two DNA extraction methods for 16S rRNA gene diversity profiling and pathogen detection. We aimed to assess which combination of DNA extraction and 16S rRNA hypervariable region enables identification of the greatest bacterial diversity, whilst minimising bias, and providing the greatest capacity for the identification of Borrelia spp.ResultsWe collected Australian endemic ticks (Bothriocroton undatum), isolated DNA from equal tick halves using two commercial DNA extraction methods and sequenced samples using V1-V3 and V3-V4 16S rRNA gene diversity profiling assays. Two distinct Borrelia spp. operational taxonomic units (OTUs) were detected using the V1-V3 16S rRNA hypervariable region and matching Borrelia spp. sequences were obtained using a conventional nested-PCR. The tick 16S rRNA gene diversity profile was dominated by Rickettsia spp. (98–99%), while the remaining OTUs belonged to Proteobacteria (51–81%), Actinobacteria (6–30%) and Firmicutes (2–7%). Multiple comparisons tests demonstrated biases in each of the DNA extraction kits towards different bacterial taxa.ConclusionsTwo distinct Borrelia species belonging to the reptile-associated Borrelia group were identified. Our results show that the method of DNA extraction can promote bias in the final microbiota identified. We determined an optimal DNA extraction method and 16S rRNA gene diversity profile assay that maximises detection of Borrelia species.
Highlights
Knowledge on the capacity of Australian ticks to carry Borrelia species is currently limited or missing
Bisecting ticks for comparison of DNA extraction methods Prior to DNA isolation, individual ticks were surface sterilised [28]; 1 min submerged in 3% hydrogen peroxide (H2O2), 30 s submerged in 70% (w/v) ethanol and 2 min submerged in phosphate buffered saline (PBS, pH = 7.4)
The direct comparison of two DNA extraction protocols, each subjected to two 16S rRNA gene diversity profiling assays, demonstrated bias associated with routine DNA extraction methods
Summary
Knowledge on the capacity of Australian ticks to carry Borrelia species is currently limited or missing. To evaluate the potential of ticks to carry bacterial pathogens and their DNA, it is imperative to have a robust workflow that maximises recovery of bacterial DNA within ticks in order to enable accurate identification. We aimed to assess which combination of DNA extraction and 16S rRNA hypervariable region enables identification of the greatest bacterial diversity, whilst minimising bias, and providing the greatest capacity for the identification of Borrelia spp. Baseline data and a consensus of potentially pathogenic bacteria in Australian ticks are imperative to initiate inquiry into the existence of the bacterial causality of human disease. To evaluate the potential of ticks to carry bacterial pathogens, bacterial DNA, it is imperative to obtain a high quality and quantity of bacterial DNA in order to enable unbiased tick surveys. There is a need to determine the bias of DNA extraction methods on a homogenous tick population
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