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A MULTIPLEX REAL-TIME PCR ASSAY AND VERIFICATION OF COMMERCIAL REAL-TIME PCR KIT FOR DERMATOPHYTE DIAGNOSTICS
2017 (English)Independent thesis Basic level (professional degree), 10 credits / 15 HE creditsStudent thesis
Abstract [en]

Dermatophytes are the most common of fungal pathogens that cause superficial infections in humans and animals. Fungi, including dermatophytes infection have a high incidence in the world. About 70 percent of the population is infected at some point in life.The study was divided into two main parts in order to establish real-time PCR technique for diagnosis of human dermatophytes in nail. The first part was to verify a commercial multiplex real-time PCR kit and the other part was to develop and optimize a new in-house multiplex real-time PCR.During verification of the commercial kit, 20 nails samples were used to compare sensitivity and specificity of a commercial assay with the conventional PCR assay run in the routine laboratory. No significant differences between the two methods were obtained; showing 100 percent specificity and sensitivity.During optimization of the recently developed multiplex real-time PCR in-house assay for pan-dermatophyte, T. interdigitale and T. rubrum, primer- and probe concentrations were chosen to 300nM and 200 nM, respectively, in a total volume of 25 μl. The real-time multiplex PCR was internally controlled by introducing a universal exogenous qPCR positive control for TaqMan-assays.In conclusion, the use of the new real-time PCR assay for the dermatophytes in nails directly in clinical samples will significantly increase detection rate and thereby reduce time compared to the assay used in the routine laboratory. However, further optimization of the new multiplex real-time PCR assay is needed.

Place, publisher, year, edition, pages
2017.
Keywords [en]
In-house- PCR, Color Compensation, Takjon, Gelelktrofores, IPC.
National Category
Biomedical Laboratory Science/Technology
Identifiers
URN: urn:nbn:se:uu:diva-349087OAI: oai:DiVA.org:uu-349087DiVA, id: diva2:1199406
Educational program
Biomedical Laboratory Science Programme; Biomedical Laboratory Science Programme
Examiners
Available from: 2018-04-20 Created: 2018-04-20 Last updated: 2018-04-20Bibliographically approved

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CiteExportLink to record
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