Rapid Detection of Enteroviruses in Small Volumes of Natural Waters by Real-Time Quantitative Reverse Transcriptase PCR

Abstract

Despite viral contamination of recreational waters, only bacterial, not viral, indicators are monitored routinely, due to a lack of rapid and cost-effective assays. We used negatively charged filters to capture enteroviruses from seawater and freshwater. Viral RNA was extracted using a commercial kit, and the viruses were quantified by real-time quantitative reverse transcriptase PCR (qRT-PCR). Poliovirus (6.6 to 330,000 virus particles/ml) was added to samples from watersheds in Los Angeles, California, and analysis showed that with 50-ml samples, a cellulose acetate/nitrate (HA) filter yielded final recovery of 51% (r2= 0.99) in fresh water and 23% (r2= 0.90) in seawater. However, for additions of low levels of virus (more likely to represent field samples; <10(4) enterovirus particles/ml), the recovery was lower and more variable, with HA being best in freshwater (17%, r2= 0.97) and the type GF/F glass filter having higher average recovery in seawater (GF/F, 17%; r2= 0.93; HA 12%, r2= 0.87). The optimized method was used with 1-liter field samples from two very different freshwater "creeks" that drain into Santa Monica Bay, California: Topanga Creek (TC), a relatively pristine mountain creek, and Ballona Creek (BC), a concrete-lined urban storm drain. One TC site out of 10 and 2 BC sites out of 7 tested significantly positive for enteroviruses, with higher enterovirus concentrations in BC than in TC (ca. 10 to 25 versus 1 equivalent enterovirus particle/ml). The presented filtration-qRT-PCR approach is fast (<8 h from sampling to results), sensitive, and cost efficient and is promising for monitoring viral contamination in environmental water samples.