High Resolution Melting Assay: a powerful method usable for species differentiation as well as for detection of polymorphisms associated with anthelmintic resistance?
|High Resolution Melting Assay: účinná metoda použitelná pro druhovou diferenciaci i pro detekci polymorfismů spojených s anthelmintickou rezistencí?
|Fakulta / Pracoviště MU
|High Resolution Melting Assay (HRMA) is post-PCR method which enables to reveal the genetic variation in DNA samples without the necessity of subsequent sequencing. Sequential changes (down to single nucleotide polymorphisms) are detected in amplified sequences according to their disassociation behavior. In our study, we employed a qPCR-HRMA to differentiate two of the pathogenic nematodes of ruminants – Haemonchus contortus and Ashworthius sidemi at the species level. The amplification and subsequent melting assay were specifically targeting the polymorphisms in two fragments of internal transcribed spacer 1 (ITS1) sequence. For the evaluation of melting process, a difference plot representing the most transparent expression of species-specific matrix curves were used. The occurrence of resistance in helminth species against various classes of anthelmintics has been described in a number of publications. It can be the result of a different mechanisms, such as genetic changes in the drug target genes (e.g. point mutations in ß-tubulin gene associated with benzimidazole resistance in Haemonchus contortus). These single nucleotide polymorphisms offer a means of detecting the presence of resistance using the HRMA. We show that qPCR-HRMA is rapid and reliable method which can be used not just to assign individual parasites taxa but it has also considerable potential for the detection of sequential changes associated with anthelmintic resistance.