Abstract：
COVID-19 is an ongoing public health threat worldwide driven by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Wastewater surveillance has emerged as a complementary tool to clinical surveillance to control the COVID-19 pandemic. With the emergence of new variants of SARS-CoV-2, accumulated mutations that occurred in the SARS-CoV-2 genome raise new challenges for RT-qPCR diagnosis used in wastewater surveillance. There is a pressing need to develop refined methods for modifying primer/probes to better detect these emerging variants in wastewater. Here, we exemplified this process by focusing on the Omicron variants, for which we have developed and validated a modified detection method. We first modified the primers/probe mismatches of three assays commonly used in wastewater surveillance according to in silico analysis results for the mutations of 882 sequences collected during the fifth-wave outbreak in Hong Kong, and then evaluated them alongside the seven original assays. The results showed that five of seven original assays had better sensitivity for detecting Omicron variants, with the limits of detection (LoDs) ranging from 1.53 to 2.76 copies/μL. UCDC-N1 and Charité-E sets had poor performances, having LoDs higher than 10 copies/μL and false-positive/false-negative results in wastewater testing, probably due to the mismatch and demonstrating the need for modification of primer/probe sequences. The modified assays exhibited higher sensitivity and specificity, along with better reproducibility in detecting 81 wastewater samples. In addition, the sequencing results of six wastewater samples by Illumina also validated the presence of mismatches in the primer/probe binding sites of the three assays. This study highlights the importance of re-configuration of the primer-probe sets and refinements for the sequences to ensure the diagnostic effectiveness of RT-qPCR detection.
摘要：
COVID-19是由严重急性呼吸道综合症冠状病毒2(SARS-CoV-2)驱动的全球持续的公共卫生威胁。废水监测已成为一种补充临床监测的新工具，以控制COVID-19大流行。随着SARS-CoV-2新变体的出现，SARS-CoV-2基因组中发生的累积突变对废水监测中使用的RT-qPCR诊断提出了新的挑战。迫切需要开发用于修饰引物/探针的精制方法以更好地检测废水中的这些新兴变体。这里,我们通过关注Omicron变体来举例说明这个过程，为此，我们开发并验证了一种改进的检测方法。我们首先根据香港第五波爆发期间收集的882个序列的突变的计算机模拟分析结果，修改了废水监测中常用的三种检测方法的引物/探针错配，然后与七个原始测定一起评估它们。结果表明，七个原始测定法中有五个对检测Omicron变体具有更好的灵敏度，检测限（LoD）范围为1.53至2.76拷贝/μL。UCDC-N1和Charité-E套装表现不佳，Lods高于10拷贝/μL和废水测试中的假阳性/假阴性结果，可能是由于错配和证明需要修饰引物/探针序列。修改后的检测方法显示出更高的灵敏度和特异性，在检测81个废水样品中具有更好的重现性。此外,Illumina对六个废水样品的测序结果也验证了三个测定的引物/探针结合位点中错配的存在。这项研究强调了引物-探针组的重新配置和序列的改进以确保RT-qPCR检测的诊断有效性的重要性。
