Applicability of integrated cell culture quantitative PCR (ICC-qPCR) for the detection of infectious adenovirus type 2 in UV disinfection studies

Abstract

Practical difficulties of the traditional adenovirus infectivity assay such as intensive labor requirements and longer turnaround period limit the direct use of adenovirus as a testing microorganism for systematic, comprehensive disinfection studies. In this study, we attempted to validate the applicability of integrated cell culture quantitative PCR (ICC-qPCR) as an alternative to the traditional cell culture method with human adenovirus type 2 (HAdV2) in a low-pressure UV disinfection study and to further optimize the procedures of ICC-qPCR for 24-well plate format. The relatively high stability of the hexon gene of HAdV2 was observed after exposure to UV radiation, resulting in a maximum gene copy reduction of 0.5 log₁₀ at 280 mJ cm⁻². Two-day post-inoculation incubation period and a maximum spiking level of 10⁵ MPN mL⁻¹ were selected as optimum conditions of ICC-qPCR with the tested HAdV2. An approximate 1:1 correlation of virus quantities by the traditional and ICC-qPCR cell culture based methods suggested that ICC-qPCR is a satisfactory alternative for practical application in HAdV2 disinfection studies. ICC-qPCR results, coupled with a first-order kinetic model (i.e., the inactivation rate constant of 0.0232 cm² mJ⁻¹), showed that an UV dose of 172 mJ cm⁻² achieved a 4-log inactivation credit for HAdV2. This estimate is comparable to other studies with HAdV2 and other adenovirus respiratory types. The newly optimized ICC-qPCR shows much promise for further study on its applicability of other slow replicating viruses in disinfection studies.

Keywords:

• Integrated cell culture quantitative PCR
• adenovirus type 2
• UV disinfection

Acknowledgments

We thank Jenifer Jones and Jonathan Popovici for technical assistance.

Funding

The United States Environmental Protection Agency, through its Office of Research and Development, has funded and managed the research described herein. This work has been subjected to the agency's administrative review and has been approved for external publication. Any opinions expressed in this paper are those of the authors and do not necessarily reflect the views of the agency; therefore, no official endorsement should be inferred. Any mention of trade names or commercial products does not constitute endorsement or recommendation for use.