Abstract
High resolution melting analysis (HRMA) is a single-tube method, which can be carried out rapidly as an additional step following real-time quantitative PCR (qPCR). The method enables the differentiation of genetic variation (down to single nucleotide polymorphisms) in amplified DNA fragments without sequencing. HRMA has previously been adopted to determine variability in the amplified genes of a number of organisms. However, only one work to date has focused on pathogenic parasites–nematodes from the genus Trichinella. In this study, we employed a qPCR-HRMA assay specifically targeting two sequential gene fragments–cytochrome c oxidase subunit I (COI) and expansion segment V (ESV), in order to differentiate 37 single L1 muscle larvae samples of eight Trichinella species. We show that qPCR-HRMA based on the mitochondrial COI gene allows differentiation between the sequences of PCR products of the same length. This simple, rapid and reliable method can be used to identify at the species level single larvae of eight Trichinella taxa.
Highlights
Zoonotic cosmopolitan nematodes of the genus Trichinella are causative agents of human trichinellosis, a serious human disease[1], which has been documented in 55 countries around the world[2]
A quantitative PCR (qPCR) assay in combination with High resolution melting analysis (HRMA) has been developed for detection of polymorphisms in Trichinella ESV20, resulting in the genotyping of four species–T. spiralis, T. nativa, T. britovi, and T. pseudospiralis
We developed a single-tube qPCR-HRMA method for reliable molecular species determination based on the polymorphism of the c oxidase subunit I (COI) gene and genomic DNA (gDNA) isolated from a single muscle larva of eight Trichinella species
Summary
Zoonotic cosmopolitan nematodes of the genus Trichinella are causative agents of human trichinellosis, a serious human disease[1], which has been documented in 55 countries around the world[2]. High resolution melting analysis (HRMA) was originally intended for genotyping, mutation scanning, and sequence matching, it might be suitable for species identification, since the melting profile of a PCR product and the shape of the HRM species-specific matrix curves depend on GC content, length, and nucleotide sequence[16]. In last years, this approach became frequently used for a various pathogens identification, including parasites and microorganisms. It was found that variations between the repeat sequences derived from a single isolate (intra-isolate) were higher than between isolates (inter-isolate) of a given parasite species, which led to the generation of non-overlapping HRM species-specific matrix curves
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