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A Host-Specific Blocking Primer Combined with Optimal DNA Extraction Improves the Detection Capability of a Metabarcoding Protocol for Canine Vector-Borne Bacteria

1
Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, Victoria 3050, Australia
2
Bayer Animal Health GmbH, 51373 Leverkusen, Germany
3
Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand
*
Author to whom correspondence should be addressed.
Pathogens 2020, 9(4), 258; https://doi.org/10.3390/pathogens9040258
Received: 12 March 2020 / Revised: 31 March 2020 / Accepted: 31 March 2020 / Published: 1 April 2020
Bacterial canine vector-borne diseases are responsible for some of the most life-threatening conditions of dogs in the tropics and are typically poorly researched with some presenting a zoonotic risk to cohabiting people. Next-generation sequencing based methodologies have been demonstrated to accurately characterise a diverse range of vector-borne bacteria in dogs, whilst also proving to be more sensitive than conventional PCR techniques. We report two improvements to a previously developed metabarcoding tool that increased the sensitivity and diversity of vector-borne bacteria detected from canine blood. Firstly, we developed and tested a canine-specific blocking primer that prevents cross-reactivity of bacterial primer amplification on abundant canine mitochondrial sequences. Use of our blocking primer increased the number of canine vector-borne infections detected (five more Ehrlichia canis and three more Anaplasma platys infections) and increased the diversity of bacterial sequences found. Secondly, the DNA extraction kit employed can have a significant effect on the bacterial community characterised. Therefore, we compared four different DNA extraction kits finding the Qiagen DNeasy Blood and Tissue Kit to be superior for detection of blood-borne bacteria, identifying nine more A. platys, two more E. canis, one more Mycoplasma haemocanis infection and more putative bacterial pathogens than the lowest performing kit. View Full-Text
Keywords: canine vector-borne disease; blocking primers; blood DNA extraction; next-generation sequencing; kit contaminant bacteria canine vector-borne disease; blocking primers; blood DNA extraction; next-generation sequencing; kit contaminant bacteria
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Huggins, L.G.; Koehler, A.V.; Schunack, B.; Inpankaew, T.; Traub, R.J. A Host-Specific Blocking Primer Combined with Optimal DNA Extraction Improves the Detection Capability of a Metabarcoding Protocol for Canine Vector-Borne Bacteria. Pathogens 2020, 9, 258.

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