Citation:

Ryu, H., K. Schrantz, N. Brinkman, AND L. Boczek. 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. JOURNAL OF VIROLOGICAL METHODS. Elsevier Science Ltd, New York, NY, 258:35-40, (2018).

Impact/Purpose:

Due to enteroviruses' occurrence in source water and limited information available regarding their inactivation by drinking water treatment practices, enteroviruses have been listed on all iterations of the U.S. Environmental Protection Agency’s Contaminant Candidate Lists (CCL) 1-4. The tissue culture based method has been the gold standard for the detection of infectious viruses. However, the tissue culture assay is time-consuming, expensive and labor intensive, resulting in a limited number of data points that consistently present challenges for generating inactivation rate constants of target human enteric viruses in water disinfection studies. Furthermore, the CPE produced after infection from multiple types of viruses has similar visual effects and therefore cannot detect the identity of the infectious virus. These disadvantages, inherent to the method, present critical barriers in assessing the efficacy of disinfection applications against human enteric viruses. To overcome some of these hurdles, tissue/cell culture based methods have been integrated with rapid molecular-based detection methods and have been successfully applied in disinfection studies of human adenoviruses and enteroviruses. Of particular interest, Mayer et al. (2010) reported the simultaneous quantification of two serotypes of Enterovirus B (coxsackievirus B6 and echovirus 12) and one serotype of Enterovirus C (poliovirus 1) using a singular tissue culture line with three serotype specific reverse transcriptase quantitative PCR (RTqPCR) assays. Although Mayer et al. (2010) could simultaneously detect three serotypes of enteroviruses, only 2 of the 4 species relevant to human health are represented. This study builds upon previous work to develop an integrated cell culture-RTqPCR (ICC-RTqPCR) method for the simultaneous detection of a broad range of enteroviruses (i.e., multiple species) relevant to human health. Furthermore, sample throughput was increased by optimizing the procedures of ICC-RTqPCR with 24-well cell culture trays followed by 96-well DNA extraction format to provide more data points, and we investigated the ICC-RTqPCR applicability in disinfection studies.

Description:

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 singular cell culture system coupled with four RTqPCR assays to detect infectious serotypes that represent the four human-related species of 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 four tested assays. A comparison of ICC-RTqPCR between the individual enterovirus serotypes and a mixture of all 4 viruses resulted in an approximate 1:1 correlation, demonstrating a lack of competition during incubation in cell culture and RTqPCR. The demonstrated capacity to differentiate individual viruses from the mixed virus culture provides the multiplex detection strategy that can be used for a variety of applications, including virus disinfection studies. This high-throughput, multiplex approach delivers the practical benefits of time- and cost-saving, which would allow the collection of more data leading to more statistically sound data sets for direct comparison of four enterovirus inactivation rates.

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