Optimizing virus inactivation methods for molecular detection techniques: Implications for viral protein and RNA measurements

Abstract

To facilitate the development of effective viral detection techniques, a positive control material is required for validating their quantitative performance. Inactivated viruses serve as viable control materials, as they can be handled without the constraints of biohazard safety facilities. However, inactivation alters the structure of viral component molecules, necessitating the selection of inactivation methods that have minimal effects on the target molecules relevant to molecular detection techniques. Only a limited number of studies have investigated inactivation methods to produce viral control materials. Therefore, the aim of this study was to investigate various virus inactivation methods and evaluate their impact on molecular detection techniques, with a specific focus on viral proteins and RNA. We evaluated the effects of ultraviolet (UV) irradiation, heat, beta-propiolactone (BPL), hydrogen peroxide (H₂O₂), and perchloric acid (HClO₄) inactivation methods to identify the most effective technique and its optimal conditions. Enzyme-linked immunosorbent assay (ELISA) and reverse transcription-digital polymerase chain reaction (RT-dPCR) were employed as model assays to assess the effects of these treatments on protein and RNA measurements. Among the evaluated methods, UV and heat treatments demonstrated minimal interference with ELISA, while heat treatment had the least impact on RT-dPCR measurements. Consequently, our findings revealed that heat inactivation holds the potential for producing inactivated viruses that can be effectively used in molecular detection techniques targeting both viral protein and RNA.

Keywords: ELISA; Influenza virus; RT-dPCR; Virus inactivation.