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 21st century and was more rece...
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Format: | Article |
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Public Library of Science (PLoS)
2022-01-01
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Series: | PLoS ONE |
Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9678315/?tool=EBI |
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author | Chandler C. Roe Jennifer Urbanz Candace Auten Guilherme G. Verocai Kelly Upshaw-Bia Olivia Holiday Crystal Hepp Jason W. Sahl |
author_facet | Chandler C. Roe Jennifer Urbanz Candace Auten Guilherme G. Verocai Kelly Upshaw-Bia Olivia Holiday Crystal Hepp Jason W. Sahl |
author_sort | Chandler C. Roe |
collection | DOAJ |
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 21st 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. |
first_indexed | 2024-04-11T15:52:46Z |
format | Article |
id | doaj.art-b29957b0dd33444bb9f1c0235bd62a9d |
institution | Directory Open Access Journal |
issn | 1932-6203 |
language | English |
last_indexed | 2024-04-11T15:52:46Z |
publishDate | 2022-01-01 |
publisher | Public Library of Science (PLoS) |
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series | PLoS ONE |
spelling | doaj.art-b29957b0dd33444bb9f1c0235bd62a9d2022-12-22T04:15:16ZengPublic Library of Science (PLoS)PLoS ONE1932-62032022-01-011711LupiQuant: A real-time PCR based assay for determining host-to-parasite DNA ratios of Onchocerca lupi and host Canis lupus from onchocercosis samplesChandler C. RoeJennifer UrbanzCandace AutenGuilherme G. VerocaiKelly Upshaw-BiaOlivia HolidayCrystal HeppJason W. SahlOnchocerca 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 21st 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.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9678315/?tool=EBI |
spellingShingle | Chandler C. Roe Jennifer Urbanz Candace Auten Guilherme G. Verocai Kelly Upshaw-Bia Olivia Holiday Crystal Hepp Jason W. Sahl LupiQuant: A real-time PCR based assay for determining host-to-parasite DNA ratios of Onchocerca lupi and host Canis lupus from onchocercosis samples PLoS ONE |
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_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_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_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 |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9678315/?tool=EBI |
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