Scabies is a contagious parasitic skin disease caused by the mite Sarcoptes scabiei, which affects more than 200 million people worldwide. Despite its prevalence, diagnosis remains challenging because microscopic examination of skin scrapings from patients with suspected infection, which is considered the diagnostic gold standard, is often limited by the low number of mites present in the samples. The aim of this master's thesis was to optimise and validate a real time polymerase chain reaction (PCR) assay for the detection of S. scabiei DNA in skin samples from patients in order to provide more reliable diagnostics of scabies. Two real time PCR assays were evaluated, one targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and the other targeting SSR6 tandem repeat. Optimization involved adjustment of primer and probe concentrations, resulting in high reaction efficiency. Validation was performed in accordance with ISO 15189:2022 and IVDR 2017/746 and included assessment of repeatability, reproducibility, analytical sensitivity, analytical specificity, as well as diagnostic sensitivity and specificity. The assay targeting the cox1 gene showed higher analytical sensitivity, whereas the assay targeting the SSR6 tandem repeat demonstrated better reproducibility. Diagnostic sensitivity and specificity were determined by testing 207 microscopically examined skin scraping samples. Compared to microscopy, real time PCR produced no false-positive results, while it detected 94.44 % of microscopy positive samples.
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