PROGETTAZIONE E VALUTAZIONE DI UN APPROCCIO DI METABARCODING PER IL RILEVAMENTO DI PARASSITI INTESTINALI EUCARIOTI

The incidence of parasitic intestinal infections is increasing worldwide, becoming of clinical concern for the human health. Nevertheless, clinicians continue to diagnose them with poorly sensitive and non-specific procedures, like the microscopic examination of faecal samples. Although several molecular methods have been developed in the last decade, allowing increasing the diagnostic precision, they still have some drawbacks that limit their application in diagnostics. With these premises, in this thesis we describe a feasible DNA metabarcoding approach to detect eukaryotic intestinal parasites in human faecal samples. To achieve this objective, we firstly tested in silico the amplification capability of several literature-available primer pairs targeting the 18S rRNA gene, and selected those able to identify the broadest spectrum of human intestinal parasites. Then, we tuned the experimental conditions to profitably extract total DNA from formalin-fixed stool and to increase the amplification efficiency. We used the selected primer pairs to amplify the metabarcodes of interest, and subsequently we sequenced the obtained samples on the Illumina MiSeq platform. Ultimately, we developed a general bioinformatics pipeline to analyse sequenced amplicons and to perform taxonomical identification of micro-eukaryotes. Overall, this work could be a promising starting point for developing a clinically relevant DNA metabarcoding diagnostic technique.