Differentiation of Eight Trichinella Species Using a High Resolution Melting Assay.
|Fakulta / Pracoviště MU|
|Popis||Cosmopolitan nematodes of the genus Trichinella are foodborne parasites with the high impact on animal trade and food safety. A broad range of carnivore and omnivore animals have been reported to be hosts of these parasites, including the economically important domestic pig. The representatives of the genus Trichinella are also causative agents of human trichinellosis, a serious human disease, which has been documented at least in 55 countries around the world. Based on zoogeographical, epidemiological and molecular characters, there are 12 taxa recognised in the genus Trichinella. According to ability of muscle larvae to encapsulate in host muscle tissues or not, two main clades (encapsulated and non-encapsulated) are recognized. Despite this difference in capsule formation, there are no clear morphological features useful for species differentiation. In this study, we adopted a high resolution melting analysis, a single-tube method, which can be carried out as an additional step following a real-time quantitative PCR. This method enables the distinguishing of genetic variation (down to single nucleotide polymorphisms) in amplified DNA fragments without necessity of subsequent sequencing. Our approach differentiating capacity was evaluated using eight Trichinella species(T1, T2, T3, T4, T5, T7, T10 and T11), 37 samples of genomic DNA isolated from single muscle larvae were analyzed. The assay was based on polymorphisms in 240 bp long fragment of the cytochrome c oxidase subunit I gene. This mitochondrial gene shows high sequence divergence even among closely allied species. For the evaluation of melting process have been used difference plot, which represents the most transparent form of expression of species-specific matrix curves. We show that high resolution melting analysis is rapid and reliable method that can be used to identify single larvae of eight Trichinella species. In the future we envisage that Trichinella species-specific matrix curves could be automatically evaluated using the computer application.|