Assessment of RT-qPCR Assays for Ebolavirus Detection

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Title: Assessment of RT-qPCR Assays for Ebolavirus Detection
Alternative title: Ebolavirus-RT-qPCR-testien vertailututkimus
Author: Kaloinen, Minttu
Other contributor: Helsingin yliopisto, Lääketieteellinen tiedekunta
University of Helsinki, Faculty of Medicine
Helsingfors universitet, Medicinska fakulteten
Publisher: Helsingin yliopisto
Date: 2020
Language: eng
Thesis level: master's thesis
Abstract: Since the discovery of Marburgvirus (MARV) in 1967, filoviruses have been identified as the causative agent for several outbreaks of hemorrhagic fever in equatorial Africa. High morbidity and mortality rates characterize especially Ebola virus disease (EVD) when Zaire ebolavirus (EBOV) is implicated. In Dec 2013, on outbreak of unprecedented magnitude started in West Africa, demanding international attention, aid and measures to restrict the spread of the epidemic. In Europe, the Ebola MoDERN APPROCHES FOR BEDSIDE RAPID DIAGNOSTICS (Ebola MoDRAD) project was set up to promote fast and accurate diagnosis of EVD. As part of this project, in this study six RT-qPCR assays (primer and probe sets or commercial kits) were compared to discover the best suitable for future development of field-deployable diagnostic test to be used in epidemics. Out of these methods, four are specific to ZEBOV NP (nucleoprotein) gene: assays by Weidmann et al., Trombley et al., Huang et al. and Clonit quanty ZEBOV Fast, and two multiplex assays targeting the L (RNA-dependent RNA polymerase) gene of several ebolaviruses (and marburgviruses): RealStar® Filovirus Screen RT-PCR Kit 1.0 by Altona Diagnostics as well as the assay by Jääskeläinen et al., which has subsequently been modified to include several ebolaviruses and Marburg marburvirus. The sample panel included in vitro produced RNA controls, inactivated whole viruses and negative controls. The analytical sensitivity of the assays was determined by Probit analysis for limit of detection (LOD) and the intra-assay repeatibility by calculating the cycle threshold coefficient variation percentage. All methods showed good specificity for their targets, yet the performance of the Clonit and Altona test kits was dependent on sample matrix. The golden standard assay by Trombley et al. was very sensitive, but the intra-assay covariance was also high (up to 13%). The assay by Jääskeläinen et al. was chosen for future development for the best overall reliability and the ability to detect several filoviruses.
Subject: ebolavirus
virus diagnostics

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