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 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. © 2016 pilotte et al.