Background: Isolation, contact tracing and restrictions on social movement are being globally implemented to prevent and control onward spread of SARS-CoV-2, even though the infection risk modelled on RNA detection by RT-qPCR remains biased as viral shedding and infectivity are not discerned . Thus, we aimed to develop a rapid viability RT-qPCR procedure to infer SARS-CoV-2 infectivity in clinical specimens and environmental samples .
Methods: We screened monoazide dyes and platinum compounds as viability molecular markers on five SARS-CoV-2 RNA targets . A platinum chloride-based viability RT-qPCR was then optimized using genomic RNA, and inactivated SARS-CoV-2 particles inoculated in buffer, stool, and urine . Our results were finally validated in nasopharyngeal swabs from persons who tested positive for COVID-19 and in wastewater samples positive for SARS-CoV-2 RNA . Findings: We established a rapid viability RT-qPCR that selectively detects potentially infectious SARS-CoV-2 particles in complex matrices . In particular, the confirmed positivity of nasopharyngeal swabs following the viability procedure suggests their potential infectivity, while the complete prevention of amplification in wastewater indicated either non-infectious particles or free RNA . Interpretation: The viability RT-qPCR approach provides a more accurate ascertainment of the infectious viruses detection and it may complement analyses to foster risk-based investigations for the prevention and control of new or re-occurring outbreaks with a broad application spectrum . Fundings: This work was supported by Spanish Scientific Research Council (CSIC), Generalitat Valenciana, and MICINN co-founded by AEI/FEDER, UE.