Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencing
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, w...
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| Format: | Article |
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BMC
2021-01-01
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| Series: | Microbiome |
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| Online Access: | https://doi.org/10.1186/s40168-020-00939-1 |
| _version_ | 1818364113898700800 |
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| author | Briana R. Flaherty Joel Barratt Meredith Lane Eldin Talundzic Richard S. Bradbury |
| author_facet | Briana R. Flaherty Joel Barratt Meredith Lane Eldin Talundzic Richard S. Bradbury |
| author_sort | Briana R. Flaherty |
| collection | DOAJ |
| 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 |
| first_indexed | 2024-12-13T21:59:13Z |
| format | Article |
| id | doaj.art-ef234d9f48354e73ba910e648d04d022 |
| institution | Directory Open Access Journal |
| issn | 2049-2618 |
| language | English |
| last_indexed | 2024-12-13T21:59:13Z |
| publishDate | 2021-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbiome |
| spelling | doaj.art-ef234d9f48354e73ba910e648d04d0222022-12-21T23:30:04ZengBMCMicrobiome2049-26182021-01-019111910.1186/s40168-020-00939-1Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencingBriana R. Flaherty0Joel Barratt1Meredith Lane2Eldin Talundzic3Richard S. Bradbury4Parasitic Diseases Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and PreventionParasitic Diseases Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and PreventionParasitic Diseases Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and PreventionMalaria Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and PreventionParasitic Diseases Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and PreventionAbstract 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 Abstracthttps://doi.org/10.1186/s40168-020-00939-1Molecular parasitologyAmplicon sequencingBlood microbiotaParasite biodiversityParasite detectionMolecular diagnosis |
| spellingShingle | Briana R. Flaherty Joel Barratt Meredith Lane Eldin Talundzic Richard S. Bradbury Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencing Microbiome Molecular parasitology Amplicon sequencing Blood microbiota Parasite biodiversity Parasite detection Molecular diagnosis |
| title | Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencing |
| title_full | Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencing |
| title_fullStr | Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencing |
| title_full_unstemmed | Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencing |
| title_short | Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencing |
| title_sort | sensitive universal detection of blood parasites by selective pathogen dna enrichment and deep amplicon sequencing |
| topic | Molecular parasitology Amplicon sequencing Blood microbiota Parasite biodiversity Parasite detection Molecular diagnosis |
| url | https://doi.org/10.1186/s40168-020-00939-1 |
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