Effect of azo dye treatment on the detection of human (OC43) coronavirus surrogate and SARS-CoV-2 viability based on an in-house photoactivator device

Abstract

Innovative detection methods for discriminating between infectious and non-infectious viruses have been developed to determine the risk of transmission, quarantine policy, and medical treatment. This study evaluated the efficacy of viability quantitative reverse transcription-polymerase chain reaction (vRT-qPCR) to indicate human coronavirus surrogate strain OC43 (HCoV-OC43) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viability based on azo dye treatment combined with our in-house photoactivator device. The factors affecting azo dye efficacy were optimized in tests with free viral RNA and virus suspension. A propidium monoazide (PMAxx) had a better performance than ethidium monoazide bromide (EMA) in the exclusion of false positive PCR signals, and its activity correlated with an increasing dye concentration. Although 50 µM PMAxx could completely quench amplification of free viral RNA at a concentration of 108 copies/µl, the highest dose of 2 mM PMAxx was only sufficient for a complete suppression of inactivated virus suspension at 102 pfu/100 µl. Additionally, various techniques of dye combinations, surfactant cotreatment, and double light exposure were tested to improve the result. Only the combination of 1 mM PMAxx and 10 µM EMA showed a comparable result to 2 mM PMAxx. The other evaluated conditions had a deleterious effect on the active virus. Despite an incomplete amplification inhibition, an observed quantification cycle (Cq) value difference larger than 8 between untreated virus and inactivated virus with intact RNA and compromised capsid might suggest viral infectivity. Therefore, this research provides vRT-qPCR data in discerning the infectious status of enveloped SARS-CoV-2 and its surrogate virus.