Real-time allelic assays of SARS-CoV-2 variants to enhance sewage surveillance

• Published in: Water research (Oxford) 2022-07, Vol.220, p.118686-118686, Article 118686
• Main Authors: Xu, Xiaoqing, Deng, Yu, Ding, Jiahui, Zheng, Xiawan, Li, Shuxian, Liu, Lei, Chui, Ho-kwong, Poon, Leo L.M., Zhang, Tong
• Format: Article
• Language: English
• Subjects: Allele-specific RT-qPCR; Alleles; COVID-19; Humans; Real-time; SARS-CoV-2; SARS-CoV-2 - genetics; Sewage; Sewage surveillance; Variants
• ISSN: 0043-1354; 1879-2448; 1879-2448
• DOI: 10.1016/j.watres.2022.118686

•Eight novel assays with high predictive value for eight variants.•Near real-time monitoring for emerging variants in the community.•Reliable quantification for mutation sites even in the sewage having variants mixture.•High correlation between clinical sequencing results and sewage variant signals.•A hierarchical detection workflow for flexible deployment of the method. To effectively control the ongoing outbreaks of fast-spreading SARS-CoV-2 variants, there is an urgent need to add rapid variant detection and discrimination methods to the existing sewage surveillance systems established worldwide. We designed eight assays based on allele-specific RT-qPCR for real-time allelic discrimination of eight SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, Omicron, Lambda, Mu, and Kappa) in sewage. In silico analysis of the designed assays for identifying SARS-CoV-2 variants using more than four million SARS-CoV-2 variant sequences yielded ∼100% specificity and >90% sensitivity. All assays could sensitively discriminate and quantify target variants at levels as low as 10 viral RNA copy/µL with minimal cross-reactivity to the corresponding nontarget genotypes, even for sewage samples containing mixtures of SARS-CoV-2 variants with differential abundances. Integration of this method into the routine sewage surveillance in Hong Kong successfully identified the Beta variant in a community sewage. Complete concordance was observed between the results of viral whole-genome sequencing and those of our novel assays in sewage samples that contained exclusively the Delta variant discharged by a clinically diagnosed COVID-19 patient living in a quarantine hotel. Our assays in this method also provided real-time discrimination of the newly emerging Omicron variant in sewage two days prior to clinical test results in another quarantine hotel in Hong Kong. These novel allelic discrimination assays offer a rapid, sensitive, and specific way for detecting multiple SARS-CoV-2 variants in sewage and can be directly integrated into the existing sewage surveillance systems. [Display omitted]