Long-range quantitative PCR for determining inactivation of adenovirus 2 by ultraviolet light

Abstract

An extra-long-range quantitative PCR (LR-qPCR) method was developed for estimating genome damage to adenovirus 2 caused by UV irradiation. The objective was to use LR-qPCR as a rapid method to determine adenovirus UV inactivation. The LR-qPCR consisted of two steps: a long-range PCR (up to 10kb fragment) and a real-time, quantitative (q) PCR for quantifying the products of the first PCR. We evaluated LR-qPCR with adenovirus irradiated with medium-pressure (MP, polychromatic emission) and low-pressure (LP, 254nm) mercury vapour lamps and compared results with cell culture infectivity. Using LR-qPCR, a fragment of 6kb estimated DNA damage in a linear relationship to doses between 0 and 20mJcm(-2) , and a 1-kb fragment related linearly to doses between 20 and 100mJcm(-2) . The LR-qPCR results for the 6-kb fragment were similar to infectivity assays results for adenovirus exposed to MP UV. For adenovirus irradiated with LP lamps, LR-qPCR results for the shorter fragment size (1kb) were similar to reduction in viral infectivity. No difference was observed between 10 and 6kb LR-qPCR results. The LR-qPCR can be used as a tool for estimating DNA damage caused by UV in adenovirus. The LR-qPCR results were related to reduction in viral infectivity. The use of LR-qPCR to determine DNA damage and estimate inactivation of adenovirus 2 from UV disinfection allows for same-day results compared with >7days required for cell culture. This accelerates adenovirus inactivation results for the water industry where adenovirus is used as a representative virus for crediting UV systems. This PCR approach provides a framework that can be used for other viral viability assays using the inhibition of amplification of viral nucleic acid after pretreatments, such as propidium monoazide, and for cellular biology studies of DNA damage.