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Real-Time PCR Assay for Detection of a New Simulant for Poxvirus Biothreat Agents▿

机译:用于痘病毒生物威胁剂的新型模拟物的实时PCR检测▿

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摘要

Research and financial efforts spent on biodefense technologies highlight the current concern for biothreat event preparedness. Nonhazardous but relevant “simulant” microorganisms are typically used to simplify technological developments, testing, and staff training. The bacteriophage MS2, a small RNA virus, is classically used as the reference simulant for biothreat viruses within the biodefense community. However, variola virus, considered a major threat, displays very different features (size, envelope, and double-stranded DNA genome). The size parameter is critical for aerosol sampling, detection, and protection/filtration technologies. Therefore, a panel of relevant simulants should be used to cover the diversity of biothreat agents. Thus, we investigated a new virus model, the Cydia pomonella granulovirus (baculovirus), which is currently used as a biopesticide. It displays a size similar to that of poxviruses, is enveloped, and contains double-stranded DNA. To provide a molecular tool to detect and quantify this model virus, we developed an assay based on real-time PCR, with a limit of detection ranging from roughly 10 to a few tens of target copies per μl according to the sample matrix. The specificity of the assay against a large panel of potential cross-reactive microorganisms was checked, and the suitability of the assay for environmental samples, especially aerosol studies, was determined. In conclusion, we suggest that our PCR assay allows Cydia pomonella granulovirus to be used as a simulant for poxviruses. This assay may also be useful for environmental or crop treatment studies.
机译:在生物防御技术上进行的研究和财政投入突显了当前对生物威胁事件准备的关注。无害但相关的“模拟”微生物通常用于简化技术开发,测试和人员培训。噬菌体MS2是一种小RNA病毒,通常被用作生物防御领域内生物威胁病毒的参考模拟物。但是,被认为是主要威胁的天花病毒显示出非常不同的特征(大小,包膜和双链DNA基因组)。尺寸参数对于气溶胶采样,检测和保护/过滤技术至关重要。因此,应使用一组相关的模拟物来涵盖生物威胁因子的多样性。因此,我们研究了一种新的病毒模型,即沙门氏假单胞菌颗粒病毒(杆状病毒),目前已用作生物农药。它显示出与痘病毒相似的大小,被包裹并且包含双链DNA。为了提供检测和定量此模型病毒的分子工具,我们开发了一种基于实时PCR的检测方法,根据样品基质,检测极限范围为每μl大约10到几十个目标拷贝。检查了针对大量潜在的交叉反应微生物的测定的特异性,并确定了测定对环境样品特别是气溶胶研究的适用性。总而言之,我们建议我们的PCR检测可以使Cydia pomonella granulovirus用作痘病毒的模拟物。该测定对于环境或作物处理研究也可能有用。

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