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Optimization of Legionella diagnostics by developing a multiplex real-time PCR assay for simultaneous detection of Legionella pneumophila and Legionella species
2017 (English)Independent thesis Basic level (professional degree), 10 credits / 15 HE creditsStudent thesis
Abstract [en]

The genus Legionella are intracellular organisms causing infection in the lower respiratory tracts and are accountable for global outbreaks of Legionnaires' disease. Legionella exists in various aquatic environments i.e. water systems in hospital buildings. Immunosuppressed patients are particularly vulnerable, considering the relatively high mortality rate within hospital-acquired pneumonia (5-20 %). The aim of this study was to optimize the current Legionella diagnostics by comparing different extraction methods and three real-time PCR assays for detecting L. species and L. pneumophila. Material used in this study consisted of nine clinical specimens collected from the respiratory tract from inpatients at the Uppsala University Hospital and surrounding regions, ten clinical samples from Quality Control for Molecular Diagnostics and reference bacterial cultures from Culture Collection University of Gothenburg. Extraction methods were tested with different pretreatment procedures. Different concentrations of primers and probes were combined to optimize the sensitivity and specificity of the method. Phocine herpesvirus-1 served as an inhibition control within the multiplex PCR assay. The results demonstrated 90 % and 100 % agreement when comparing current method with the commercial kit and the multiplex PCR assay, respectively. Sensitivity and specificity were improved using primer concentrations above 0.5 μM with 0.2 μM probe. Using Phocine herpesvirus-1 as an inhibition control showed successful detection in the multiplex PCR assay, although it had an impact on the detection of L. pneumophila and L. species. This indicates competition of reagents within the reaction, thus further optimization is required to improve the multiplex PCR assay.

Place, publisher, year, edition, pages
2017.
Keywords [en]
Legionnaires’ disease, in-house PCR, Phocine Herpesvirus-1, TaqMan probe, ssrA gene
National Category
Biomedical Laboratory Science/Technology
Identifiers
URN: urn:nbn:se:uu:diva-349080OAI: oai:DiVA.org:uu-349080DiVA, id: diva2:1199392
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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