Virology Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Frederick, MD, 21702-5011, USA.
Quality Assurance & Regulatory Compliance Office, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Frederick, MD, 21702-5011, USA.
Virol J. 2017 Nov 3;14(1):210. doi: 10.1186/s12985-017-0880-8.
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.
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.
The assay had a lower limit of quantitation of 50 genome copies/5 uL sample, upper limit of quantitation of 5 × 10 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.
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.
1980 年，天花病毒在自然界中被根除，天花病毒的病原体，被限制在两个实验室中，一个位于俄罗斯（莫斯科），后来转移到西伯利亚的 Vector，另一个位于美国（亚特兰大疾病控制中心）。在成功根除天花病毒后不久，公众停止了接种疫苗，导致人群对天花病毒的免疫敏感性逐渐增加。由于天花病毒有可能被故意重新引入，以及其他致病性痘病毒的出现，因此非常需要开发医学对策来治疗痘病毒病。美国食品和药物管理局（FDA）很可能需要“动物规则”来批准这些干预措施。因此，需要开发相关的动物模型和相关的支持检测方法，以应对监管审查。
美国陆军传染病研究所（USAMRIID）的研究人员开发了一种用于检测正痘病毒的优化实时 PCR 检测方法。为了支持将用于支持美国 FDA 批准医疗对策的动物研究，该检测方法旨在定量检测非人类灵长类动物（食蟹猴）血液基质中的痘病毒基因组 DNA，作为病毒血症的测量。本文描述了该方法的验证过程，包括从用 EDTA 抗凝的全血中提取 DNA，以通过评估检测方法和/或检测方法组件的精密度、准确性、标准曲线、特异性、稳健性和稳定性来获得和定量检测猴痘基因组。
该检测方法的定量下限为 50 个基因组拷贝/5 uL 样本，定量上限为 5×10 GC/5uL 样本，检测下限为 2.5 个基因组拷贝/5uL 样本。该检测方法对正痘病毒具有特异性。检测到基质效应，表明存在与目标 DNA 一起被提取的 PCR 抑制剂。
该检测方法已被验证用于定量检测食蟹猴血液中的猴痘病毒载量。该检测方法已经并将继续支持向 FDA 提交申请，以批准用于天花的抗病毒疗法。
