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The emergence of the SARS-CoV-2 variant BA.2.86.1 raised a considerable concern, due to the large number of potentially virulent mutations. In this study, we developed a novel assay that specifically detects variant BA.2.86.1, and used it to screen environmental samples from wastewater treatment sites across Israel. By using a multiplex assay that included a general SARS-CoV-2 reaction, together with the BA.2.86.1-specific reaction and a control reaction, we quantified the absolute number of viral copies in each sample and its relative abundance, compared with the total copy number of circulating SARS-CoV-2. Evaluation of the new reactions showed that they are both sensitive and specific, detecting down to four copies per reaction, and maintain specificity in the presence of Omicron variants BA.1, 2 and 4 RNA. Examination of 279 samples from 30 wastewater collection sites during August-September 2023 showed that 35 samples (12.5 %) were positive, from 18 sites. Quantitative analysis of the samples showed that the relative abundance of variant BA.2.86.1 with respect to the total viral load of SARS-CoV-2 was very low and consisted between 0.01 % and 0.6 % of the total SARS-CoV-2 circulation. This study demonstrates the importance of combining wastewater surveillance with the development of specialized diagnostic assays, when clinical testing is insufficient. This approach may be useful for timely response by public health authorities in future outbreaks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)