One Assay, Nine Targets: Advancing Viral Surveillance with Multiplex RT-ddPCR

Viral infections continue to pose a major global health challenge, driven by factors such as population growth, migration, and environmental change, all of which contribute to the emergence and reemergence of infectious viruses. Advances in technology now enable the detection of multiple targets from a limited sample volume; however, few studies have fully leveraged these capabilities. In this study, we developed and analytically validated a highly sensitive and specific 9-plex one-step RT-ddPCR assay for the detection of high-risk viruses, including SARS-CoV-2 (N1 and N2 genes), Influenza A and B, Respiratory Syncytial Virus, Hepatitis A and E, along with both endogenous and exogenous controls. Initial validation was conducted using synthetic DNA, followed by application to 38 wastewater samples─complex and heterogeneous matrices that often harbor multiple viral targets. The assay demonstrated excellent analytical performance in terms of sensitivity, linearity, specificity, and reproducibility with detection limits ranging from 1.4 to 2.9 copies/μL depending on the viral target. A direct comparison with singleplex ddPCR assays revealed high concordance (Mann-Whitney test, <i>p</i> > 0.1), indicating no statistically significant differences and highlighting the efficiency of the multiplex format. To the best of our knowledge, this is the first study to simultaneously quantify nine targets in a single RT-ddPCR reaction. The developed assay shows a strong potential for application across various sample types, including wastewater.