COVID-19 wastewater surveillance has gained widespread acceptance to monitor community infection trends. Wastewater samples primarily differ from clinical samples by having low viral concentrations due to dilution, and high levels of PCR inhibitors. Therefore, wastewater samples should be processed by appropriately designed and optimized molecular workflows to accurately quantify targets. Digital PCR has shown to be more sensitive and resilient to environmental matrix inhibition. However, most SARS-CoV-2 assays have been designed for clinical use on RT-qPCR instruments, then adopted to digital PCR platforms. But it is unknown whether clinical RT-qPCR assays are adequate to use on digital PCR platforms. Here we designed an N and E gene multiplex (ddCoV_N and ddCoV_E) specifically for RT-ddPCR and benchmarked them against the nCoV_N2 and E_Sarbeco assays. ddCoV_N and ddCoV_E have equivalent limits of detections and wastewater sample concentrations to NCoV_N2 and E_Sarbeco but showed improved signal-to-noise ratios that eased interpretation and ability to multiplex. From GISAID downloaded unique sequences analyzed, 2.12% and 0.83% present a mismatch or would not be detected by the used primer/probe combination for the ddCoV_N and ddCoV_E, respectively.

中文翻译：

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重新设计用于废水 RT-ddPCR 的 SARS-CoV-2 临床 RT-qPCR 检测方法

COVID-19 废水监测已获得广泛接受，可用于监测社区感染趋势。废水样品与临床样品的主要区别在于由于稀释而具有低病毒浓度和高水平的 PCR 抑制剂。因此，废水样品应通过适当设计和优化的分子工作流程进行处理，以准确量化目标。数字 PCR 已显示对环境基质抑制更敏感和更有弹性。然而，大多数 SARS-CoV-2 检测方法都是为 RT-qPCR 仪器的临床应用而设计的，然后被用于数字 PCR 平台。但尚不清楚临床 RT-qPCR 检测是否足以在数字 PCR 平台上使用。在这里，我们设计了一个专门用于 RT-ddPCR 的 N 和 E 基因复合物（ddCoV_N 和 ddCoV_E），并将它们与 nCoV_N2 和 E_Sarbeco 检测进行了基准测试。ddCoV_N 和 ddCoV_E 的检测限和废水样品浓度与 NCoV_N2 和 E_Sarbeco 相当，但显示出改进的信噪比，从而简化了解释和复用能力。从 GISAID 下载的独特序列分析中，2.12% 和 0.83% 分别存在不匹配或不会被 ddCoV_N 和 ddCoV_E 使用的引物/探针组合检测到。