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Capsid Integrity qPCR—An Azo-Dye Based and Culture-Independent Approach to Estimate Adenovirus Infectivity after Disinfection and in the Aquatic Environment

1
Singapore Centre for Life Science Engineering, Nanyang Technological University, Singapore 637551, Singapore
2
School of Public Health, University of Alberta, Edmonton, AB T6G 2G7, Canada
3
Department of Environmental Science and Engineering, Gilling’s School of Global Public Health, University of North Carolina, Chapel Hill, NC 27516, USA
4
Department of Hygiene, Social and Environmental Medicine, Ruhr-University Bochum, 44801 Bochum, Germany
5
Federal State Agency for Consumer & Health Protection Rhineland-Palatinate, 56068 Koblenz, Germany
*
Author to whom correspondence should be addressed.
Water 2019, 11(6), 1196; https://doi.org/10.3390/w11061196
Received: 7 May 2019 / Revised: 30 May 2019 / Accepted: 6 June 2019 / Published: 8 June 2019
(This article belongs to the Special Issue Water Quality Impacts of Contaminant Transport and Transformation)
Recreational, reclaimed and drinking source waters worldwide are under increasing anthropogenic pressure, and often contain waterborne enteric bacterial, protozoan, and viral pathogens originating from non-point source fecal contamination. Recently, the capsid integrity (ci)-qPCR, utilizing the azo-dyes propidium monoazide (PMA) or ethidium monoazide (EMA), has been shown to reduce false-positive signals under laboratory conditions as well as in food safety applications, thus improving the qPCR estimation of virions of public health significance. The compatibility of two widely used human adenovirus (HAdV) qPCR protocols was evaluated with the addition of a PMA/EMA pretreatment using a range of spiked and environmental samples. Stock suspensions of HAdV were inactivated using heat, UV, and chlorine before being quantified by cell culture, qPCR, and ci-qPCR. Apparent inactivation of virions was detected for heat and chlorine treated HAdV while there was no significant difference between ci-qPCR and qPCR protocols after disinfection by UV. In a follow-up comparative analysis under more complex matrix conditions, 51 surface and 24 wastewater samples pre/post UV treatment were assessed for enteric waterborne HAdV to evaluate the ability of ci-qPCR to reduce the number of false-positive results when compared to conventional qPCR and cell culture. Azo-dye pretreatment of non-UV inactivated samples was shown to improve the ability of molecular HAdV quantification by reducing signals from virions with an accessible genome, thereby increasing the relevance of qPCR results for public health purposes, particularly suited to resource-limited low and middle-income settings. View Full-Text
Keywords: capsid integrity qPCR; human adenovirus; surface water; viral infectivity; water quality indicator capsid integrity qPCR; human adenovirus; surface water; viral infectivity; water quality indicator
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MDPI and ACS Style

Leifels, M.; Shoults, D.; Wiedemeyer, A.; Ashbolt, N.J.; Sozzi, E.; Hagemeier, A.; Jurzik, L. Capsid Integrity qPCR—An Azo-Dye Based and Culture-Independent Approach to Estimate Adenovirus Infectivity after Disinfection and in the Aquatic Environment. Water 2019, 11, 1196.

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