LupiQuant: A real-time PCR based assay for determining host-to-parasite DNA ratios of Onchocerca lupi and host Canis lupus from onchocercosis samples
Onchocerca lupi is a filarial nematode that causes ocular onchocercosis in canines globally including North America and areas of Europe, North Africa, and the Middle East. Reported incidence of this parasite in canines has continued to steadily escalate since the early 21 st century and was more rec...
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crplos:10.1371/journal.pone.0276916 2024-05-19T07:38:45+00:00 LupiQuant: A real-time PCR based assay for determining host-to-parasite DNA ratios of Onchocerca lupi and host Canis lupus from onchocercosis samples Roe, Chandler C. Urbanz, Jennifer Auten, Candace Verocai, Guilherme G. Upshaw-Bia, Kelly Holiday, Olivia Hepp, Crystal Sahl, Jason W. Portugaliza, Harvie P. Pacific Southwest Center of Excellence in Vector-Borne Diseases 2022 http://dx.doi.org/10.1371/journal.pone.0276916 https://dx.plos.org/10.1371/journal.pone.0276916 en eng Public Library of Science (PLoS) http://creativecommons.org/licenses/by/4.0/ PLOS ONE volume 17, issue 11, page e0276916 ISSN 1932-6203 journal-article 2022 crplos https://doi.org/10.1371/journal.pone.0276916 2024-05-01T06:58:48Z Onchocerca lupi is a filarial nematode that causes ocular onchocercosis in canines globally including North America and areas of Europe, North Africa, and the Middle East. Reported incidence of this parasite in canines has continued to steadily escalate since the early 21 st century and was more recently documented in humans. Whole genome sequencing (WGS) of this parasite can provide insight into gene content, provide novel surveillance targets, and elucidate the origin and range expansion. However, past attempts of whole genome sequencing of other Onchocerca species reported a substantial portion of their data unusable due to the variable over-abundance of host DNA in samples. Here, we have developed a method to determine the host-to-parasite DNA ratio using a quantitative PCR (qPCR) approach that relies on two standard plasmids each of which contains a single copy gene specific to the parasite genus Onchocerca (major body wall myosin gene, myosin) or a single copy gene specific to the canine host (polycystin-1 precursor, pkd1 ). These plasmid standards were used to determine the copy number of the myosin and pkd1 genes within a sample to calculate the ratio of parasite and host DNA. Furthermore, whole genome sequence (WGS) data for three O . lupi isolates were consistent with our host-to-parasite DNA ratio results. Our study demonstrates, despite unified DNA extraction methods, variable quantities of host DNA within any one sample which will likely affect downstream WGS applications. Our quantification assay of host-to-parasite genome copy number provides a robust and accurate method of assessing canine host DNA load in an O . lupi specimen that will allow informed sample selection for WGS. This study has also provided the first whole genome draft sequence for this species. This approach is also useful for future focused WGS studies of other parasites. Article in Journal/Newspaper Canis lupus PLOS PLOS ONE 17 11 e0276916 |
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language |
English |
description |
Onchocerca lupi is a filarial nematode that causes ocular onchocercosis in canines globally including North America and areas of Europe, North Africa, and the Middle East. Reported incidence of this parasite in canines has continued to steadily escalate since the early 21 st century and was more recently documented in humans. Whole genome sequencing (WGS) of this parasite can provide insight into gene content, provide novel surveillance targets, and elucidate the origin and range expansion. However, past attempts of whole genome sequencing of other Onchocerca species reported a substantial portion of their data unusable due to the variable over-abundance of host DNA in samples. Here, we have developed a method to determine the host-to-parasite DNA ratio using a quantitative PCR (qPCR) approach that relies on two standard plasmids each of which contains a single copy gene specific to the parasite genus Onchocerca (major body wall myosin gene, myosin) or a single copy gene specific to the canine host (polycystin-1 precursor, pkd1 ). These plasmid standards were used to determine the copy number of the myosin and pkd1 genes within a sample to calculate the ratio of parasite and host DNA. Furthermore, whole genome sequence (WGS) data for three O . lupi isolates were consistent with our host-to-parasite DNA ratio results. Our study demonstrates, despite unified DNA extraction methods, variable quantities of host DNA within any one sample which will likely affect downstream WGS applications. Our quantification assay of host-to-parasite genome copy number provides a robust and accurate method of assessing canine host DNA load in an O . lupi specimen that will allow informed sample selection for WGS. This study has also provided the first whole genome draft sequence for this species. This approach is also useful for future focused WGS studies of other parasites. |
author2 |
Portugaliza, Harvie P. Pacific Southwest Center of Excellence in Vector-Borne Diseases |
format |
Article in Journal/Newspaper |
author |
Roe, Chandler C. Urbanz, Jennifer Auten, Candace Verocai, Guilherme G. Upshaw-Bia, Kelly Holiday, Olivia Hepp, Crystal Sahl, Jason W. |
spellingShingle |
Roe, Chandler C. Urbanz, Jennifer Auten, Candace Verocai, Guilherme G. Upshaw-Bia, Kelly Holiday, Olivia Hepp, Crystal Sahl, Jason W. LupiQuant: A real-time PCR based assay for determining host-to-parasite DNA ratios of Onchocerca lupi and host Canis lupus from onchocercosis samples |
author_facet |
Roe, Chandler C. Urbanz, Jennifer Auten, Candace Verocai, Guilherme G. Upshaw-Bia, Kelly Holiday, Olivia Hepp, Crystal Sahl, Jason W. |
author_sort |
Roe, Chandler C. |
title |
LupiQuant: A real-time PCR based assay for determining host-to-parasite DNA ratios of Onchocerca lupi and host Canis lupus from onchocercosis samples |
title_short |
LupiQuant: A real-time PCR based assay for determining host-to-parasite DNA ratios of Onchocerca lupi and host Canis lupus from onchocercosis samples |
title_full |
LupiQuant: A real-time PCR based assay for determining host-to-parasite DNA ratios of Onchocerca lupi and host Canis lupus from onchocercosis samples |
title_fullStr |
LupiQuant: A real-time PCR based assay for determining host-to-parasite DNA ratios of Onchocerca lupi and host Canis lupus from onchocercosis samples |
title_full_unstemmed |
LupiQuant: A real-time PCR based assay for determining host-to-parasite DNA ratios of Onchocerca lupi and host Canis lupus from onchocercosis samples |
title_sort |
lupiquant: a real-time pcr based assay for determining host-to-parasite dna ratios of onchocerca lupi and host canis lupus from onchocercosis samples |
publisher |
Public Library of Science (PLoS) |
publishDate |
2022 |
url |
http://dx.doi.org/10.1371/journal.pone.0276916 https://dx.plos.org/10.1371/journal.pone.0276916 |
genre |
Canis lupus |
genre_facet |
Canis lupus |
op_source |
PLOS ONE volume 17, issue 11, page e0276916 ISSN 1932-6203 |
op_rights |
http://creativecommons.org/licenses/by/4.0/ |
op_doi |
https://doi.org/10.1371/journal.pone.0276916 |
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PLOS ONE |
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17 |
container_issue |
11 |
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e0276916 |
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1799478235036123136 |