Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design.

BACKGROUND:The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world's most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measure...

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Published in:PLOS Neglected Tropical Diseases
Main Authors: Nils Pilotte, Marina Papaiakovou, Jessica R Grant, Lou Ann Bierwert, Stacey Llewellyn, James S McCarthy, Steven A Williams
Format: Article in Journal/Newspaper
Language:English
Published: Public Library of Science (PLoS) 2016
Subjects:
Online Access:https://doi.org/10.1371/journal.pntd.0004578
https://doaj.org/article/f09e395bcb0d4066a4142f43deacdc40
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spelling ftdoajarticles:oai:doaj.org/article:f09e395bcb0d4066a4142f43deacdc40 2023-05-15T15:13:53+02:00 Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design. Nils Pilotte Marina Papaiakovou Jessica R Grant Lou Ann Bierwert Stacey Llewellyn James S McCarthy Steven A Williams 2016-03-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0004578 https://doaj.org/article/f09e395bcb0d4066a4142f43deacdc40 EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC4814118?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0004578 https://doaj.org/article/f09e395bcb0d4066a4142f43deacdc40 PLoS Neglected Tropical Diseases, Vol 10, Iss 3, p e0004578 (2016) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2016 ftdoajarticles https://doi.org/10.1371/journal.pntd.0004578 2022-12-31T08:58:02Z BACKGROUND:The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world's most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays. METHODOLOGY/PRINCIPAL FINDINGS:Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay. CONCLUSIONS/SIGNIFICANCE:The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other eukaryotic pathogens. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 10 3 e0004578
institution Open Polar
collection Directory of Open Access Journals: DOAJ Articles
op_collection_id ftdoajarticles
language English
topic Arctic medicine. Tropical medicine
RC955-962
Public aspects of medicine
RA1-1270
spellingShingle Arctic medicine. Tropical medicine
RC955-962
Public aspects of medicine
RA1-1270
Nils Pilotte
Marina Papaiakovou
Jessica R Grant
Lou Ann Bierwert
Stacey Llewellyn
James S McCarthy
Steven A Williams
Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design.
topic_facet Arctic medicine. Tropical medicine
RC955-962
Public aspects of medicine
RA1-1270
description BACKGROUND:The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world's most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays. METHODOLOGY/PRINCIPAL FINDINGS:Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay. CONCLUSIONS/SIGNIFICANCE:The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other eukaryotic pathogens.
format Article in Journal/Newspaper
author Nils Pilotte
Marina Papaiakovou
Jessica R Grant
Lou Ann Bierwert
Stacey Llewellyn
James S McCarthy
Steven A Williams
author_facet Nils Pilotte
Marina Papaiakovou
Jessica R Grant
Lou Ann Bierwert
Stacey Llewellyn
James S McCarthy
Steven A Williams
author_sort Nils Pilotte
title Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design.
title_short Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design.
title_full Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design.
title_fullStr Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design.
title_full_unstemmed Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design.
title_sort improved pcr-based detection of soil transmitted helminth infections using a next-generation sequencing approach to assay design.
publisher Public Library of Science (PLoS)
publishDate 2016
url https://doi.org/10.1371/journal.pntd.0004578
https://doaj.org/article/f09e395bcb0d4066a4142f43deacdc40
geographic Arctic
geographic_facet Arctic
genre Arctic
genre_facet Arctic
op_source PLoS Neglected Tropical Diseases, Vol 10, Iss 3, p e0004578 (2016)
op_relation http://europepmc.org/articles/PMC4814118?pdf=render
https://doaj.org/toc/1935-2727
https://doaj.org/toc/1935-2735
1935-2727
1935-2735
doi:10.1371/journal.pntd.0004578
https://doaj.org/article/f09e395bcb0d4066a4142f43deacdc40
op_doi https://doi.org/10.1371/journal.pntd.0004578
container_title PLOS Neglected Tropical Diseases
container_volume 10
container_issue 3
container_start_page e0004578
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