title: Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design.
abstract: 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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Department of Biological Sciences, Smith College, Northampton, Massachusetts, United States of America.|Molecular and Cellular Biology Program, University of Massachusetts Amherst, Amherst, Massachusetts, United States of America.
authors.forename: Nils
authors.initials: N
authors.lastname: Pilotte
authors.affiliation: Department of Biological Sciences, Smith College, Northampton, Massachusetts, United States of America.
authors.forename: Marina
authors.initials: M
authors.lastname: Papaiakovou
authors.affiliation: Department of Biological Sciences, Smith College, Northampton, Massachusetts, United States of America.
authors.forename: Jessica R
authors.initials: JR
authors.lastname: Grant
authors.affiliation: Department of Biological Sciences, Smith College, Northampton, Massachusetts, United States of America.
authors.forename: Lou Ann
authors.initials: LA
authors.lastname: Bierwert
authors.affiliation: QIMR Berghofer Medical Research Institute, Brisbane, Australia.
authors.forename: Stacey
authors.initials: S
authors.lastname: Llewellyn
authors.affiliation: QIMR Berghofer Medical Research Institute, Brisbane, Australia.
authors.forename: James S
authors.initials: JS
authors.lastname: McCarthy
authors.affiliation: Department of Biological Sciences, Smith College, Northampton, Massachusetts, United States of America.|Molecular and Cellular Biology Program, University of Massachusetts Amherst, Amherst, Massachusetts, United States of America.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Williams
mesh_terms: D000818:Animals; D018509:DNA, Helminth / Q000302:isolation & purification*; D006373:Helminthiasis / Q000175:diagnosis* / Q000469:parasitology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012987:Soil / Q000469:parasitology*
chemical_list: D018509:DNA, Helminth; D012987:Soil