Direct PCR with the CDC 2019 SARS-CoV-2 assay: optimization for limited-resource settings.
Direct PCR with the CDC 2019 SARS-CoV-2 assay: optimization for limited-resource settings.
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CDC 2019 SARS-CoV-2检测的直接PCR：针对有限资源环境的优化。
DOI：
10.1038/s41598-022-15356-7
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发表时间：
2022-07-11
影响因子：
4.6
通讯作者：
Adams, Nicholas M
中科院分区：
文献类型：
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作者：
Victoriano, Christia M;Pask, Megan E;Malofsky, Nicole A;Seegmiller, Adam;Simmons, Steve;Schmitz, Jonathan E;Haselton, Frederick R;Adams, Nicholas M
基于PCR的诊断通常需要在扩增之前从患者样本中提取核酸。正如COVID-19疫情初期所强调，在需求量大且试剂供应有限的时期，提取步骤可能难以扩大规模。放弃提取步骤，我们之前报道了广泛使用的CDC COVID-19检测试剂的N1引物/探针组在将病毒转运培养基（VTM）直接接种到qRT-PCR反应中时保持了较高的分类灵敏度（95%）和特异性（100%）。相比之下，N2集表现出突出的Ct延迟和低灵敏度（33%），而没有提取。在本研究中，我们通过结合N1/N2/RNase P多重分析并分析退火温度、VTM干扰和接种体积的影响，改进了该改良CDC检测试剂盒（特别是N2组）的性能。后两个因素对N2的性能比N1产生更显著的影响，尽管这些影响在很大程度上通过提高退火温度来克服。用41份SARS-CoV-2阳性和43份阴性临床样本对该未提取/多重方案进行了评估，证明与未修改的CDC方法相比，分类灵敏度为92.7%，特异性为100%。总体而言，这项工作提供了一种可推广的策略，以在资源有限的情况下最大限度地提高COVID-19或其他新兴病原体的检测能力。
PCR-based diagnostics generally require nucleic acid extraction from patient specimens prior to amplification. As highlighted early in the COVID-19 pandemic, extraction steps may be difficult to scale during times of massive demand and limited reagent supply. Forgoing an extraction step, we previously reported that the N1 primer/probe-set of the widespread CDC COVID-19 assay maintains high categorical sensitivity (95%) and specificity (100%) with direct inoculation of viral transport media (VTM) into qRT-PCR reactions. In contrast, the N2 set demonstrated a prominent Ct delay and low sensitivity (33%) without extraction. In the current study, we have improved the performance of this modified CDC assay (in particular the N2 set) by incorporating N1/N2/RNase P multiplexing and dissecting the effects of annealing temperature, VTM interference, and inoculum volume. The latter two factors exerted a more prominent effect on the performance of N2 than N1, although these effects were largely overcome through elevated annealing temperature. This unextracted/multiplex protocol was evaluated with 41 SARS-CoV-2 positive and 43 negative clinical samples, demonstrating a categorical sensitivity of 92.7% and specificity of 100% versus the unmodified CDC methodology. Overall, this work offers a generalizable strategy to maximize testing capabilities for COVID-19 or other emerging pathogens when resources are constrained.