Validation of a pan-orthopox real-time PCR assay for the detection and quantification of viral genomes from nonhuman primate blood
| Autor: | David Miller, Wendy Giles, Donna Hering, Chris Hartmann, Eric M. Mucker, Robert J. Fisher, John W. Huggins |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
viruses 030106 microbiology Viremia Genome Viral Real-Time Polymerase Chain Reaction Sensitivity and Specificity lcsh:Infectious and parasitic diseases 03 medical and health sciences Monkeypox Virology medicine Animals Smallpox lcsh:RC109-216 Orthopoxvirus Monkeypox virus biology United States Food and Drug Administration Methodology virus diseases medicine.disease biology.organism_classification DNA extraction United States Vaccination Macaca fascicularis Infectious Diseases DNA Viral Immunology Variola virus Viral load |
| Zdroj: | Virology Journal, Vol 14, Iss 1, Pp 1-9 (2017) Virology Journal |
| DOI: | 10.1186/s12985-017-0880-8 |
| Popis: | Background In 1980, smallpox disease was eradicated from nature and Variola virus, the etiological agent of smallpox, was confined to two laboratories, one located in Russia (Moscow) later moved to VECTOR (Novosibirsk, Siberia) and one in the United States (CDC Atlanta). Vaccinations among the general public ceased shortly after the successful eradication campaign, resulting in an increasingly immunologically susceptible population. Because of the possibility of intentional reintroduction of Variola virus and the emergence of other pathogenic poxviruses, there is a great need for the development of medical countermeasures to treat poxvirus disease. It is highly likely that the U.S. FDA “animal rule” will be necessary for regulatory approval of these interventions. Therefore, relevant animal models and the associated supporting assays will require development to stand up to regulatory scrutiny. Methods An optimized real time PCR assay for the detection of orthopoxviruses has been developed by researchers at the United States Army Research Institute of Infectious Diseases (USAMRIID). To support animal studies that will be used to support approval of medical countermeasures by the U.S. FDA, the assay was designed to quantitate poxvirus genomic DNA in a nonhuman primate (cynomolgus macaque) blood matrix as a measurement of viremia. This manuscript describes the validation of the process, including DNA extraction from whole blood anticoagulated with EDTA, for obtaining and quantitating monkeypox genomes by evaluating precision, accuracy, the standard curve, specificity, robustness and stability of the assay and/or components of the assay. Results The assay had a lower limit of quantitation of 50 genome copies/5 uL sample, upper limit of quantitation of 5 × 107 GC/5uL sample and a limit of detection of 2.5 genome copies /5uL sample. The assay was specific for orthopoxvirus. Matrix effects were detected and suggest the presence of PCR inhibitor(s) that was co-extracted with the target DNA. Conclusions The assay has been validated for the purpose of quantitating monkeypox viral load in blood from cynomolgus macaques. This assay has and will continue to support submissions to the FDA for approval of antiviral therapeutics for smallpox. Electronic supplementary material The online version of this article (10.1186/s12985-017-0880-8) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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