Ozone disinfection kinetics of poliovirus 1 determined by cell culture assay, RT-qPCR and ethidium monoazide qPCR reduction in a continuous quench-flow reactor.

Abstract

A continuous quench-flow (CQF) reactor was developed to collect samples at the reaction times of less than one second. The reactor is applied to determine ozone disinfection kinetics of poliovirus and to study whether EMA-qPCR can assess the viral infectivity after ozone disinfection. Ozone disinfection of poliovirus was conducted in the developed CQF, and the disinfection kinetics were tested in the range of 0·7-5·0s at ozone concentration of 0·08 and 0·25mgl-1 . Inactivation, damage on viral genome and damage on capsid integrity were determined by plaque assay, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and ethidium monoazide treatment coupled with RT-qPCR (EMA-qPCR), respectively. By using CQF, 2·18 and 2·76 log10 reductions were observed at the reaction time of 0·7s and ozone concentration of 0·08 and 0·25mgl-1 , respectively, followed by tailing. Ozone disinfection kinetics of poliovirus 1 were better fit by the efficiency factor Hom model than by the Chick-Watson model, or the modified Chick-Watson model. Kinetics observed were similar between RT-qPCR and EMA-qPCR assays at the reaction times of <2·0s and ozone concentrations of 0·08 and 0·25mgl-1 . At reaction times >5s, viral concentration evaluated by EMA-qPCR was reduced in comparison to stable RT-qPCR results. Both assays still underestimated the virus inactivation. The simple developed reactor can be used to investigate viral ozone disinfection kinetics and to elucidate inactivation characteristics or mechanisms at very short exposure times. The developed CQF reactor is beneficial for better understanding of virus inactivation by ozone, and the reactor can be used to better elucidate disinfection kinetics and mechanisms for future research. This work constitutes an important contribution to the existing knowledge of the application and limitation of the EMA/PMA-qPCR to assess virus infectivity after ozone disinfection.