Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencing.

Flaherty, BR; Barratt, J; Lane, M; Talundzic, E; Bradbury, RS

Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencing.

Flaherty, BR Barratt, J

Lane, M Talundzic, E Bradbury, RS

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Publisher:: BioMed Central
Publication Type:: Journal Article
Citation:: Microbiome, 2021, 9, (1), pp. 1-19
Issue Date:: 2021-01-02

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Field	Value	Language
dc.contributor.author	Flaherty, BR
dc.contributor.author	Barratt, J https://orcid.org/0000-0001-8711-2408
dc.contributor.author	Lane, M
dc.contributor.author	Talundzic, E
dc.contributor.author	Bradbury, RS
dc.date.accessioned	2022-01-14T01:28:09Z
dc.date.available	2020-10-14
dc.date.available	2022-01-14T01:28:09Z
dc.date.issued	2021-01-02
dc.identifier.citation	Microbiome, 2021, 9, (1), pp. 1-19
dc.identifier.issn	2049-2618
dc.identifier.issn	2049-2618
dc.identifier.uri	http://hdl.handle.net/10453/153098
dc.description.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. Video Abstract.
dc.format	Electronic
dc.language	eng
dc.publisher	BioMed Central
dc.relation.ispartof	Microbiome
dc.relation.isbasedon	10.1186/s40168-020-00939-1
dc.rights	info:eu-repo/semantics/restrictedAccess
dc.rights	© 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 . The Creative Commons Public Domain Dedication waiver ( ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
dc.subject	0602 Ecology, 0605 Microbiology, 1108 Medical Microbiology
dc.subject.mesh	Animals
dc.subject.mesh	Blood
dc.subject.mesh	DNA Primers
dc.subject.mesh	DNA Restriction Enzymes
dc.subject.mesh	DNA, Ribosomal
dc.subject.mesh	High-Throughput Nucleotide Sequencing
dc.subject.mesh	Humans
dc.subject.mesh	Malaria
dc.subject.mesh	Parasites
dc.subject.mesh	Polymerase Chain Reaction
dc.subject.mesh	RNA, Ribosomal, 18S
dc.subject.mesh	Animals
dc.subject.mesh	Blood
dc.subject.mesh	DNA Primers
dc.subject.mesh	DNA Restriction Enzymes
dc.subject.mesh	DNA, Ribosomal
dc.subject.mesh	High-Throughput Nucleotide Sequencing
dc.subject.mesh	Humans
dc.subject.mesh	Malaria
dc.subject.mesh	Parasites
dc.subject.mesh	Polymerase Chain Reaction
dc.subject.mesh	RNA, Ribosomal, 18S
dc.subject.mesh	Blood
dc.subject.mesh	Animals
dc.subject.mesh	Humans
dc.subject.mesh	Parasites
dc.subject.mesh	Malaria
dc.subject.mesh	DNA Restriction Enzymes
dc.subject.mesh	DNA, Ribosomal
dc.subject.mesh	RNA, Ribosomal, 18S
dc.subject.mesh	DNA Primers
dc.subject.mesh	Polymerase Chain Reaction
dc.subject.mesh	High-Throughput Nucleotide Sequencing
dc.title	Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencing.
dc.type	Journal Article
utslib.citation.volume	9
utslib.location.activity	England
utslib.for	0602 Ecology
utslib.for	0605 Microbiology
utslib.for	1108 Medical Microbiology
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Faculty of Science/School of Life Sciences
utslib.copyright.status	recently_added	*
pubs.consider-herdc	false
dc.date.updated	2022-01-14T01:28:04Z
pubs.issue	1
pubs.publication-status	Published
pubs.volume	9
utslib.citation.issue	1

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. Video Abstract.

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http://hdl.handle.net/10453/153098