- Title
- Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencing
- Creator
- Flaherty, Briana; Barratt, Joel; Lane, Meredith; Talundzic, Eldin; Bradbury, Richard
- Date
- 2021
- Type
- Text; Journal article
- Identifier
- http://researchonline.federation.edu.au/vital/access/HandleResolver/1959.17/175587
- Identifier
- vital:15005
- Identifier
-
https://doi.org/10.1186/s40168-020-00939-1
- Identifier
- ISBN:2049-2618 (ISSN)
- Abstract
- Background: Targeted amplicon deep sequencing (TADS) has enabled characterization of diverse bacterial communities, yet the application of TADS to communities of parasites has been relatively slow to advance. The greatest obstacle to this has been the genetic diversity of parasitic agents, which include helminths, protozoa, arthropods, and some acanthocephalans. Meanwhile, universal amplification of conserved loci from all parasites without amplifying host DNA has proven challenging. Pan-eukaryotic PCRs preferentially amplify the more abundant host DNA, obscuring parasite-derived reads following TADS. Flaherty et al. (2018) described a pan-parasitic TADS method involving amplification of eukaryotic 18S rDNA regions possessing restriction sites only in vertebrates. Using this method, host DNA in total DNA extracts could be selectively digested prior to PCR using restriction enzymes, thereby increasing the number of parasite-derived reads obtained following NGS. This approach showed promise though was only as sensitive as conventional PCR. Results: Here, we expand on this work by designing a second set of pan-eukaryotic primers flanking the priming sites already described, enabling nested PCR amplification of the established 18S rDNA target. This nested approach facilitated introduction of a second restriction digestion between the first and second PCR, reducing the proportional mass of amplifiable host-derived DNA while increasing the number of PCR amplification cycles. We applied this method to blood specimens containing Babesia, Plasmodium, various kinetoplastids, and filarial nematodes and confirmed its limit of detection (LOD) to be approximately 10-fold lower than previously described, falling within the range of most qPCR methods. Conclusions: The assay detects and differentiates the major malaria parasites of humans, along with several other clinically important blood parasites. This represents an important step towards a TADS-based universal parasite diagnostic (UPDx) test with a sufficient LOD for routine applications. [MediaObject not available: see fulltext.]. © 2021, The Author(s).
- Publisher
- BioMed Central Ltd
- Relation
- Microbiome Vol. 9, no. 1 (2021), p.
- Rights
- All metadata describing materials held in, or linked to, the repository is freely available under a CC0 licence
- Rights
- http://creativecommons.org/licenses/by/4.0/
- Rights
- Copyright © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
- Rights
- Open Access
- Subject
- 0602 Ecology; 0605 Microbiology; 1108 Medical Microbiology; Amplicon sequencing; Blood microbiota; Molecular diagnosis; Molecular parasitology; Parasite biodiversity; Parasite detection
- Full Text
- Reviewed
- Funder
- Funding for this work was provided by the Centers for Disease Control and Prevention Advanced Molecular Detection (AMD) initiative and the CDC’s Parasitic Diseases Branch (PDB) within the Division of Parasitic Diseases and Malaria (DPDM).
- Hits: 2181
- Visitors: 2238
- Downloads: 130
| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | SOURCE1 | Published version | 2 MB | Adobe Acrobat PDF | View Details Download |