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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

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Cunningham, Lucas ORCID: https://orcid.org/0000-0002-3017-4074, Stothard, Russell ORCID: https://orcid.org/0000-0002-9370-3420, Osei-Atweneboana, Mike, Armoo, Samuel, Verweij, Jaco J. and Adams, Emily ORCID: https://orcid.org/0000-0002-0816-2835 (2018) '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'. Parasitology, Vol 145, Issue 13, pp. 1733-1738.

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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.

Item Type: Article
Additional Information: 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)
Subjects: QU Biochemistry > Genetics > QU 550 Genetic techniques. PCR. Chromosome mapping
WC Communicable Diseases > Tropical and Parasitic Diseases > WC 800 Helminthiasis
WC Communicable Diseases > Tropical and Parasitic Diseases > WC 810 Schistosomiasis
Faculty: Department: Biological Sciences > Department of Tropical Disease Biology
Digital Object Identifer (DOI): https://doi.org/10.1017/s0031182018001361
SWORD Depositor: JISC Pubrouter
Depositing User: Stacy Murtagh
Date Deposited: 10 Sep 2018 14:46
Last Modified: 28 Feb 2019 02:02
URI: https://archive.lstmed.ac.uk/id/eprint/9266

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