Wastewater multiplex PCR amplicon sequencing revealed community transmission of SARS-CoV-2 lineages during the outbreak of infection in Chinese Mainland.

During the COVID-19, wastewater-based epidemiology (WBE) has become a powerful epidemic surveillance tool widely used worldwide. However, the development and application of this technology in Chinese Mainland are relatively lagging. Herein, we for the first time monitored the community circulation of SARS-CoV-2 lineages using WBE methods in Chinese Mainland. During the peak period of infection outbreak at the end of 2022, six precious sewage samples were collected from the manhole in the student dormitory area on Wangjiang Campus of Sichuan University. RT-qPCR revealed that the six sewage samples were all positive for SARS-CoV-2 RNA. Multiplex PCR amplicon sequencing of the sewage samples reflected the local transmission of SARS-CoV-2 variants. The results of two deconvolution methods indicate that the main virus lineages have clear evolutionary genetic correlations. Furthermore, the sampling time is consistent with the timeline of concern for these virus lineages, as well as the timeline of uploading the nucleic acid sequences from the corresponding lineages in Sichuan to the database. These results demonstrate the reliability of the sewage sequencing results. Multiplex PCR amplicon sequencing is by far the most powerful analytical tool of WBE, enabling quantitative detection of virus lineages transmission and evolution at the community level.