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.

FIG. 1.

Map of field sample locations. The light zone marked “Los Angeles” is largely urbanized, and the darker “Santa Monica Mountains” has greatly reduced population density.

FIG. 2.

qRT-PCR-estimated recovery of seeded polioviruses from freshwater samples with three filter types. The y axis indicates where the sample results occurred on the standard curve prepared from poliovirus. Lines are linear regressions forced through the origin. Panel A shows data for the entire concentration range, and panel B shows only the lower range. All data points from duplicate measurements (two assays from each extracted filter) are shown. The same poliovirus stock was used for the seeding (x axis) and for the standard curve used to calibrate the y axis, hence the slope reflects the recovery. In the poliovirus stock used there was approximately 1 PFU per 66 enterovirus particles.

FIG. 3.

qRT-PCR-estimated recovery of seeded polioviruses from seawater samples with three filter types. This figure is the same as Fig. 2, but it is for seawater samples.

FIG. 4.

Amount of RNA detected in an extract after it was subjected to two freeze-thaw cycles as a percentage of the amount of RNA detected after one freeze-thaw cycle.

FIG. 5.

Flow chart of protocol for measuring enteroviruses in natural waters, with dashed lines indicating possible locations of tests for recovery and efficiency of various steps. diam., diameter.