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Additionally, optimization of a respiratory RT-qPCR multiplex assay to detect influenza A, respiratory syncytial virus, and SARS-CoV-2 was attempted. The enteric multiplex assays successfully detected Coxsackievirus A6 in wastewater during community outbreaks of hand-foot-mouth disease. Enterovirus D68 was also successfully detected in wastewater samples (Summer\/Fall, 2022), which coincided with provincial public health reports of Enterovirus D68 cases. Attempting to optimize the respiratory multiplex assay resulted in challenges due to oligonucleotide cross-reactivity and cross-talk. Specifically, when Texas Red and FAM probes detected higher abundance targets, they interfered with the Cy5 and HEX fluorophore probes that detected lower-abundance targets. In contrast, selecting probes with Cy5\/HEX for high-abundance targets and Texas Red\/FAM for lower-abundance targets provided more robust results.<\/jats:p>","DOI":"10.2166\/wh.2025.101","type":"journal-article","created":{"date-parts":[[2025,4,15]],"date-time":"2025-04-15T17:01:33Z","timestamp":1744736493000},"page":"546-560","update-policy":"https:\/\/doi.org\/10.2166\/iwapcrossmarkpolicypage","source":"Crossref","is-referenced-by-count":1,"title":["Optimizing RT-qPCR multiplex assays for simultaneous detection of enteric and respiratory viruses in wastewater"],"prefix":"10.2166","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-4158-9014","authenticated-orcid":false,"given":"Tom\u00e1s","family":"de Melo","sequence":"first","affiliation":[{"name":"1 Faculty of Science, Ontario Tech University, 2000 Simcoe St N, Oshawa, ON L1G 0C5, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ashley","family":"Gedge","sequence":"additional","affiliation":[{"name":"1 Faculty of Science, Ontario Tech University, 2000 Simcoe St N, Oshawa, ON L1G 0C5, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Denina B. 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Ontario: Ontario Ministry of Environmental Conservation and Parks. Available at: https:\/\/files.ontario.ca\/mecp-protocol-for-analyzing-wastewater-samples-en-2022-03-23.pdf (Accessed: 6 February 2025)."},{"key":"key-10.2166\/wh.2025.101-24","unstructured":"Pasieka C. (2022) \u2018Ontario Doctors Warn There's a Surge of Hand-Foot-and-Mouth Disease in Kids and Parents\u2019, CBC News, 7 July. Toronto: Canadian Broadcasting Company News. Available at: https:\/\/www.cbc.ca\/news\/canada\/toronto\/hand-foot-mouth-disease-2022-1.6511796 (Accessed: 25 December 2024)."},{"key":"key-10.2166\/wh.2025.101-25","unstructured":"Public Health Ontario (2022) Enterovirus D68 Testing at Public Health Ontario. Toronto: Public Health Ontario. 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