Rapid detection of enteroviruses in small volumes of natural waters by real-time quantitative reverse transcriptase PCR.
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.
Fuhrman
Jed A
JA
Department of Biological Sciences and Wrigley Institute for Environmental Studies, University of Southern California, Los Angeles, CA 90089-0371, USA. fuhrman@usc.edu
Liang
Xiaolin
X
Noble
Rachel T
RT
D002482:Cellulose / Q000031:analogs & derivatives; D003101:Collodion; D004770:Enterovirus / Q000302:isolation & purification*; D005374:Filtration / Q000379:methods; D005618:Fresh Water / Q000821:virology*; D005898:Glass; D017955:Poliovirus / Q000302:isolation & purification; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012623:Seawater / Q000821:virology*; D013997:Time Factors; D014773:Virology / Q000379:methods
D012367:RNA, Viral; C005062:acetylcellulose; D002482:Cellulose; D003101:Collodion