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Developing a real-time PCR assay based on multiplex high-resolution melt-curve analysis: a pilot study in detection and discrimination of soil-transmitted helminth and schistosome species

Published online by Cambridge University Press:  28 August 2018

Lucas J. Cunningham Open the ORCID record for Lucas J. Cunningham [Opens in a new window] ,
J. Russell Stothard Open the ORCID record for J. Russell Stothard [Opens in a new window] ,
Mike Osei-Atweneboana ,
Samuel Armoo ,
Jaco J. Verweij  and
Emily R. Adams
Lucas J. Cunningham*
Affiliation:
Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK
J. Russell Stothard
Affiliation:
Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK
Mike Osei-Atweneboana
Affiliation:
Department of Environmental Biology and Health, Council for Scientific and Industrial Research – Water Research Institute, P.O. Box M 32, Accra 102001, Ghana
Samuel Armoo
Affiliation:
Department of Environmental Biology and Health, Council for Scientific and Industrial Research – Water Research Institute, P.O. Box M 32, Accra 102001, Ghana
Jaco J. Verweij
Affiliation:
Laboratory for Medical Microbiology and Immunology, Elisabeth Tweesteden, Tilburg, The Netherlands
Emily R. Adams
Affiliation:
Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK
*
Author for correspondence: Lucas J. Cunningham, E-mail: lucas.cunningham@lstmed.ac.uk
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Abstract

With the push towards control and elimination of soil-transmitted helminthiasis and schistosomiasis in low- and middle-income countries, there is a need to develop alternative diagnostic assays that complement the current in-country resources, preferably at a lower cost. Here, we describe a novel high-resolution melt (HRM) curve assay with six PCR primer pairs, designed to sub-regions of the nuclear ribosomal locus. Used within a single reaction and dye detection channel, they are able to discriminate Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Ascaris lumbricoides, Trichuris trichiuria and Schistosoma spp. by HRM curve analysis. Here we describe the primers and the results of a pilot assessment whereby the HRM assay was tested against a selection of archived fecal samples from Ghanaian children as characterized by Kato–Katz and real-time PCR analysis with species-specific TaqMan hydrolysis probes. The resulting sensitivity and specificity of the HRM was 80 and 98.6% respectively. We judge the assay to be appropriate in modestly equipped and resourced laboratories. This method provides a potentially cheaper alternative to the TaqMan method for laboratories in lower resource settings. However, the assay requires a more extensive assessment as the samples used were not representative of all target organisms.

Keywords

Disease surveillanceDNAHRMmelt-curve analysismonitoring and evaluationreal-time PCRSCHschistosomiasissoil-transmitted helminthiasisSTH
Type
Special Issue Research Article
Information
Parasitology , Volume 145 , Special Issue 13: 2017 Autumn Symposium of the British Society for Parasitology The multi-disciplinarity of parasitology: Host-parasite evolution in an ever changing world , November 2018 , pp. 1733 - 1738
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Copyright
Copyright © Cambridge University Press 2018 

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