Development and validation of an integrated cell culture-qRTPCR assay for simultaneous quantification of coxsackieviruses, echoviruses, and polioviruses in disinfection studies.
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.
Mayer
B K
BK
Arizona State University, Tempe, AZ 85287-5306, USA. Brooke.Mayer@asu.edu
Ryu
H
H
Gerrity
D
D
Abbaszadegan
M
M
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004203:Disinfection / Q000379:methods*; D004770:Enterovirus / Q000302:isolation & purification*; D029822:Enterovirus B, Human / Q000302:isolation & purification*; D007668:Kidney / Q000166:cytology; D017955:Poliovirus / Q000302:isolation & purification*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014871:Water Microbiology; D018508:Water Purification