Multiplex PCR amplicon sequencing revealed community transmission of SARS-CoV-2 lineages on the campus of Sichuan University during the outbreak of infection in Chinese Mainland at the end of 2022

During the pandemic of COVID-19, wastewater-based epidemiology has become a powerful epidemic surveillance tool widely used around the world. However, the development and application of this technology in Chinese Mainland are relatively lagging. Herein, we report the first case of community circulation of SARS-CoV-2 lineages monitored by WBE in Chinese Mainland during the infection outbreak at the end of 2022 after the comprehensive relaxation of epidemic prevention policies. During the peak period of infection, six precious sewage samples were collected from the manhole in the student dormitory area of Wangjiang Campus of Sichuan University. According to the results RT-qPCR, the six sewage samples were all positive for SARS-CoV-2 RNA. Based on multiplex PCR amplicon sequencing, the local transmission of SARS-CoV-2 variants at that time was analyzed. The results show that the main virus lineages in sewage have clear evolutionary genetic correlations. Furthermore, the sampling time is very consistent with the timeline of concern for these virus lineages and consistent with the timeline for uploading the nucleic acid sequences of the corresponding lineages in Sichuan to the database. These results demonstrate the reliability of the sequencing results of SARS-CoV-2 nucleic acid in wastewater. Multiplex PCR amplicon sequencing is by far the most powerful analytical tool of WBE, enabling quantitative monitoring of virus lineage prevalence at the community level. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by the National Natural Science Foundation of China (No. 22176133, No. 21677104). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors