Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays
| Autor: | Ruth Harvey, Kathryn S. Doris, Neil Almond, Stacey Efstathiou, Philip D. Minor, Kirsty MacLellan-Gibson, Dianna E. Wilkinson, Carolyn Nicolson, Rob Anderson, Giada Mattiuzzo, Mark Page, James Ashall |
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| Rok vydání: | 2015 |
| Předmět: |
RNA Stability
viruses lcsh:Medicine Biology medicine.disease_cause Virus VP40 medicine Humans lcsh:Science Multidisciplinary Ebola virus lcsh:R Lentivirus Virion virus diseases RNA Nucleic acid amplification technique Hemorrhagic Fever Ebola Reference Standards Ebolavirus Virology Africa Western HEK293 Cells Calibration HIV-1 Nucleic acid RNA Viral lcsh:Q RNA extraction Nucleic Acid Amplification Techniques Research Article |
| Zdroj: | PLoS ONE PLoS ONE, Vol 10, Iss 11, p e0142751 (2015) |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0142751 |
| Popis: | The 2013-present Ebola virus outbreak in Western Africa has prompted the production of many diagnostic assays, mostly based on nucleic acid amplification technologies (NAT). The calibration and performance assessment of established assays and those under evaluation requires reference materials that can be used in parallel with the clinical sample to standardise or control for every step of the procedure, from extraction to the final qualitative/quantitative result. We have developed safe and stable Ebola virus RNA reference materials by encapsidating anti sense viral RNA into HIV-1-like particles. The lentiviral particles are replication-deficient and non-infectious due to the lack of HIV-1 genes and Envelope protein. Ebola virus genes were subcloned for encapsidation into two lentiviral preparations, one containing NP-VP35-GP and the other VP40 and L RNA. Each reference material was formulated as a high-titre standard for use as a calibrator for secondary or internal standards, and a 10,000-fold lower titre preparation to serve as an in-run control. The preparations have been freeze-dried to maximise stability. These HIV-Ebola virus RNA reference materials were suitable for use with in-house and commercial quantitative RT-PCR assays and with digital RT-PCR. The HIV-Ebola virus RNA reference materials are stable at up to 37°C for two weeks, allowing the shipment of the material worldwide at ambient temperature. These results support further evaluation of the HIV-Ebola virus RNA reference materials as part of an International collaborative study for the establishment of the 1st International Standard for Ebola virus RNA. |
| Databáze: | OpenAIRE |
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