The use of High Resolution Melting analysis for reliable differentiation of trichostrongylid nematodes Haemonchus contortus and Ashworthius sidemi
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| Year of publication | 2020 |
| Type | Conference abstract |
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| Description | Infection of ruminants by helminth parasites, especially gastrointestinal nematodes, has worldwide a considerable economic and social impact. From this perspective, correct taxonomical identification is a key prerequisite of initiation of appropriate measures. However, differentiation of some nematode species according to morphological features is complicated, e.g. in the case of important haematophagous abomasal parasites, Haemonchus contortus and Ashworthius sidemi. In this study, we employed qPCR followed by High Resolution Melting (HRM) analysis as an advanced molecular method. This approach enables to detect sequence alterations by increasing temperature. Differences in melting profiles can be visualised as the fluorescence of saturating dye that is gradually disassociated from the dsDNA amplicons. As an output, a difference plot representing the most transparent expression of specific matrix curves were used. The aim of this study was to develop a fast and usable qPCR-HRM assay, which uses polymorphisms in internal transcribed spacer 1 (ITS-1) to distinguish both nematodes without the necessity of additional confirmation by electrophoretic separation and/or sequencing. Based on the specific melting curves, we identified a total of 45 specimens of adult nematodes of H. contortus and A. sidemi that came from a wide range of domestic and wild ruminant hosts that occur in the Czech Republic. We also confirmed that qPCR-HRM analysis is applicable for determination of the infective larval stages of the nematodes, which promises a significant improvement in intravital diagnostics. |
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