Optimizing RT-qPCR multiplex assays for simultaneous detection of enteric and respiratory viruses in wastewater.

This study presents the successful optimization of enteric RT-qPCR multiplex assays for detecting Norovirus GII, Enterovirus, and Coxsackievirus A6 or Enterovirus D68 in municipal wastewater samples. 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.