We describe the use of in silico approaches to improve the process of molecular assay development and reduce time and cost by utilizing available databases of whole genome pathogen sequences combined with modern bioinformatics and physical modeling tools. Well-characterized assays are needed for accurately detecting pathogens in environmental and patient samples and also for evaluation of the efficacy of a medical countermeasure that may be administered to patients. The polymerase chain reaction (PCR) remains the gold standard for pathogen detection due to the simplicity of its instrumentation, low cost of reagents, and outstanding limit of detection (LOD), sensitivity, and specificity. However, creation of such PCR assays often involves iterations of design, preliminary testing, and thorough validation with clinical isolates and testing in relevant matrices, which can be time consuming, costly, and result in suboptimal assays. Since formal validation (e.g., for Emergency Use Authorization [EUA] or Food and Drug Administration [FDA] licensure) of an infectious disease assay can be very expensive and can require extensive time of development, having a well-designed assay up front is a critical first step. Yet, many assays described in the literature utilized limited design capabilities and many initially promising assays fail the validation process, resulting in increased costs and timelines for successful product development. While the computational approaches outlined in this document by no means obviate the need for wet lab testing, they can reduce the amount of effort wasted on empirical optimization and iterative redesigns and also guide validation studies. The proposed computational approaches also result in higher performing assays with better sensitivity, specificity, and lower LOD and reduce the possibility of assay failure due to signature erosion. To provide clarity, an extensive glossary of defined terms is provided.

中文翻译：

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附录 Q：使用现代计算机方法开发微生物病原体检测分子测定的建议


我们描述了通过利用全基因组病原体序列的可用数据库结合现代生物信息学和物理建模工具，使用计算机方法来改进分子测定开发过程并减少时间和成本。需要经过充分表征的分析方法来准确检测环境和患者样本中的病原体，并评估可能对患者采取的医疗对策的有效性。聚合酶链式反应 (PCR) 因其仪器简单、试剂成本低廉以及出色的检测限 (LOD)、灵敏度和特异性，仍然是病原体检测的黄金标准。然而，此类 PCR 测定的创建通常涉及设计的迭代、初步测试、临床分离株的彻底验证以及相关基质中的测试，这可能非常耗时、成本高昂，并且会导致测定结果不理想。由于传染病检测的正式验证（例如，紧急使用授权 [EUA] 或食品药品监督管理局 [FDA] 许可）可能非常昂贵，并且可能需要大量的开发时间，因此预先设计良好的检测是一个必要的条件。关键的第一步。然而，文献中描述的许多测定方法利用了有限的设计能力，并且许多最初有希望的测定方法未能通过验证过程，导致成功产品开发的成本和时间表增加。虽然本文件中概述的计算方法决不能消除湿实验室测试的需要，但它们可以减少在经验优化和迭代重新设计上浪费的精力，并指导验证研究。 所提出的计算方法还可以实现更高性能的检测，具有更好的灵敏度、特异性和更低的 LOD，并减少由于特征侵蚀而导致检测失败的可能性。为了清晰起见，提供了广泛的术语定义表。