Abstract：
Wastewater-based surveillance can be used as a complementary method to other SARS-CoV-2 surveillance systems. It allows the emergence and spread of infections and SARS-CoV-2 variants to be monitored in time and place. This study presents an RT-ddPCR method that targets the T19I amino acid mutation in the spike protein of the SARS-CoV-2 genomes, which is specific to the BA.2 variant (omicron). The T19I assay was evaluated both in silico and in vitro for its inclusivity, sensitivity, and specificity. Moreover, wastewater samples were used as a proof of concept to monitor and quantify the emergence of the BA.2 variant from January until May 2022 in the Brussels-Capital Region which covers a population of more than 1.2 million inhabitants. The in silico analysis showed that more than 99% of the BA.2 genomes could be characterized using the T19I assay. Subsequently, the sensitivity and specificity of the T19I assay were successfully experimentally evaluated. Thanks to our specific method design, the positive signal from the mutant probe and wild-type probe of the T19I assay was measured and the proportion of genomes with the T19I mutation, characteristic of the BA.2 mutant, compared to the entire SARS-CoV-2 population was calculated. The applicability of the proposed RT-ddPCR method was evaluated to monitor and quantify the emergence of the BA.2 variant over time. To validate this assay as a proof of concept, the measurement of the proportion of a specific circulating variant with genomes containing the T19I mutation in comparison to the total viral population was carried out in wastewater samples from wastewater treatment plants in the Brussels-Capital Region in the winter and spring of 2022. This emergence and proportional increase in BA.2 genomes correspond to what was observed in the surveillance using respiratory samples; however, the emergence was observed slightly earlier, which suggests that wastewater sampling could be an early warning system and could be an interesting alternative to extensive human testing.
摘要：
基于废水的监测可以用作其他SARS-CoV-2监测系统的补充方法。它允许在时间和地点监测感染和SARS-CoV-2变体的出现和传播。本研究提出了一种RT-ddPCR方法，该方法针对SARS-CoV-2基因组的刺突蛋白中的T19I氨基酸突变，这是特定于BA.2变体（omicron）。T19I测定法在计算机和体外评估其包容性，灵敏度,和特异性。此外，废水样本被用作概念证明，以监测和量化2022年1月至5月在布鲁塞尔首都地区出现的BA.2变体，该地区人口超过120万。计算机分析显示,超过99%的BA.2基因组可以使用T19I测定来表征。随后，通过实验成功评估了T19I检测的敏感性和特异性.由于我们特定的方法设计，测量来自T19I测定的突变探针和野生型探针的阳性信号，并测量具有T19I突变的基因组的比例，BA.2突变体的特征，与整个SARS-CoV-2人群相比。评估了所提出的RT-ddPCR方法的适用性，以监测和定量BA.2变体随时间的出现。为了验证这个实验作为概念证明，2022年冬季和春季,在布鲁塞尔首都大区污水处理厂的废水样本中,对含有T19I突变基因组的特定循环变异体与总病毒种群的比例进行了测量.BA.2基因组的这种出现和成比例的增加与在使用呼吸道样本的监测中观察到的情况相对应；然而，出现的时间稍早，这表明废水采样可能是一个早期预警系统，可能是一个有趣的替代广泛的人类测试。
