Applicability of integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) for the simultaneous detection of the four human enteric enterovirus species in disinfection studies

Abstract

A newly developed integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method and its applicability in UV disinfection studies is described. This method utilizes a cell culture system coupled with four RTqPCR assays to detect four species of human enterovirus (e.g., Enterovirus A, Enterovirus B, Enterovirus C and Enterovirus D). Evaluation of the RTqPCR assays was conducted with coxsackievirus A10, echovirus 30, poliovirus 1 and enterovirus 70 and resulted in 100% specificity for the tested assays. A comparison of ICC-RTqPCR between the individual enteroviruses and a mixture of all four viruses resulted in an approximate 1:1 correlation, demonstrating a lack of competition during incubation in cell culture and RTqPCR. The simultaneous detection of multiple human enterovirus species within mixed cultures is relevant to many applications, including virus disinfection studies. This high-throughput, multiplex approach costs less in money and time. By helping with data collection, this approach will lead to more statistically sound data sets that directly compare the inactivation rates of enteroviruses tested.

Efficacy of Inactivation of Human Enteroviruses by Dual-Wavelength Germicidal Ultraviolet (UV-C) Light Emitting Diodes (LEDs)

“…Triplicate CB tests were performed with mixed stocks of four viruses. Infectious virus concentrations were determined using an integrated cell culture reverse transcriptase quantitative polymerase chain reaction (ICC-RTqPCR), as described previously [31,32]. Log 10 inactivation of enteroviruses (I) is defined by Equation (1):…”

Progress in the study of virus detection methods: The possibility of alternative methods to validate virus inactivation

“…Li et al, 2010;F. Li, Feng, Liu, Zhen, & Chen, 2009;Mahalanabis, Reynolds, Pepper, & Gerba, 2010;Mayer, Ryu, Gerrity, & Abbaszadegan, 2010;Ogorzaly et al, 2013;Reynolds, 2001Reynolds, , 2004Reynolds, Gerba, & Pepper, 1996;Rodríguez, Polston, Wu, Wu, & Sobsey, 2013;Ryu et al, 2018;Ryu, Schrantz, Brinkman, & Boczek, 2018). ICC-qPCR utilizes the host cell as an efficient tool to separate infectious and noninfectious viruses because only the living virus can inject its genome into the host cell for amplification.…”

“…Currently, ICC-qPCR has been utilized in validation studies of enteroviruses (Chapron et al, 2000;S. H. Lee et al, 2005;Mayer et al, 2010;Ogorzaly et al, 2013;Reynolds, 2001Reynolds, , 2004Reynolds et al, 1996;Ryu et al, 2018) (Chapron et al, 2000;Gerrity et al, 2008;Guo et al, 2018;Ryu et al, 2015;Ko et al, 2003;Ko et al, 2005;S. H. Lee et al, 2005;F.…”

“…Although increase in the incubation time can be utilized to increase the quantity of viral genome copies in cells to increase accuracy (Guo et al, 2018;Ryu et al, 2018), the long incubation time will impact the samples with a high inoculation concentration (Gerrity et al, 2008). A presupposition for utilizing cells to amplify infectious viruses (viral nucleic acid) that are then detected by qPCR was that the replication rate of the viral nucleic acid in the cells be constant according to the following equation:…”

“…Li et al, 2010;F. Li et al, 2009;Mahalanabis et al, 2010;Mayer et al, 2010;Ogorzaly et al, 2013;Reynolds, 2001Reynolds, , 2004Reynolds et al, 1996;Rodríguez et al, 2013;Ryu et al, 2018;Jiang et al, 2010 Abbreviations: EMA, ethidium monoazide; PMA, propidium monoazide; HCV, hepatitis C virus; PK, proteinase K; qPCR, quantitative polymerase chain reaction; UV, ultraviolet.…”