Development and validation of an integrated cell culture-qRTPCR assay for simultaneous quantification of coxsackieviruses, echoviruses, and polioviruses in disinfection studies

Abstract

This study demonstrated the applicability of integrated cell culture-quantitative RTPCR (ICC-qRTPCR) for the simultaneous quantification of coxsackievirus, echovirus, and poliovirus in disinfection studies. Buffalo green monkey cells were inoculated with a 10-fold dilution series of mixed enteroviruses and incubated prior to qRTPCR quantification. Optimal assay conditions included three post infection washes and a 24-hour post infection incubation period based on successful differentiation between infectious and noninfectious viruses and significant and consistent viral replication rates. Ultraviolet disinfection studies were performed to validate the ICC-qRTPCR assay. Using the optimized assay, three-log microbial inactivation was achieved at UV doses of 30-44, 28-42, and 28-29 mJ/cm(2) for coxsackievirus B6, echovirus 12, and poliovirus 1, respectively. These results compare favorably to side-by-side assessments using conventional cultural techniques and values previously reported in the literature. This indicates that ICC-qRTPCR is a practical alternative for the simultaneous quantification of enteroviruses in disinfection studies.