Investigation on the applicability of a long-range reverse-transcription quantitative polymerase chain reaction assay for the rapid detection of active viruses

Abstract

Abstract Background Although conventional polymerase chain reaction (PCR) methods are widely used in diagnosis, the titer of the pathogenic virus is difficult to determine based on the PCR. In our prior report, a long-range reverse-transcription quantitative PCR (LR-RT-qPCR) assay was developed to assess the titer of UV-irradiated influenza A virus (IAV) rapidly. In this research, we focused on whether the LR-RT-qPCR assay could evaluate the titer of IAV inactivated by other methods. Methods IAV was inactivated by: heating at 100 °C for periods ranging from 1 to 15 min, treating with 0.12% sodium hypochlorite for periods ranging from 3 to 30 min, or treating with 70% ethanol for periods ranging from 10 to 30 min. Fifty percent tissue culture infectious dose (TCID50) assay was performed to confirm the efficacy of the inactivation methods, followed by LR-RT-qPCR to investigate the correlation between infectivity and copy number. Results One minute heating, 3 min sodium hypochlorite treatment, or 10 min ethanol treatment was sufficient to deactivate IAV. Changes before and after the inactivations in the copy numbers on LR-RT-qPCR were significantly different among the inactivation methods. Heat-inactivation drastically decreased the copy number to below the cutoff value around 5 copies/μL after 5 min treatment. The inactivation time of heating estimated using LR-RT-qPCR was marginally higher than that determined using TCID50. However, the treatments with sodium hypochlorite or ethanol moderately and minimally affected the copy numbers obtained using LR-RT-qPCR (~ 1 digit or no copy number decrease), respectively. Conclusions In addition to good applicability in UV-irradiation previously reported, the LR-RT-qPCR method is suitable for evaluating the effect of heat-inactivation on IAV infectivity. However, minor modifications may be made and investigated in the future to reduce the time intervals with TCID50. Although this method is not applicable for the ethanol inactivation, rapid evaluation of the effects of chlorination on IAV can be determined by comparing copy numbers before and after treatment using the LR-RT-qPCR method.

Keywords

• Infectivity
• Inactivation
• Heat-inactivation
• Sodium hypochlorite
• Ethanol