Concentration and detection of caliciviruses in water samples by reverse transcription-PCR

Human caliciviruses (HuCVs) cause waterborne outbreaks of gastroenteritis. Standard indicators of a safe water supply do not adequately predict contam ination of water by viruses, including HuCVs. We developed a method to conc entrate and detect HuCVs in water samples by using a cultivable primate cal icivirus (Pan-l) as a model. Viable Pan-1 was seeded in different types of water and then filtered with a 1MDS filter, eluted with beef extract (BE), and reconcentrated by polyethylene glycol (PEG) precipitation. The viruses in the final samples were tested by plaque assay or by reverse transcriptio n (RT)-PCR following extraction of the RNA with Trizol. Pan-1 was more sens itive to high-pa treatment than poliovirus was; a pH 9.0 BE solution was fo und to recover 35% more viable Pan-1 than a pH 9.5 BE solution recovered. P an-1 was recovered from small volumes of deionized, finished, ground, and s urface waters at efficiencies of 94, 73, 67, and 64%, respectively, when sa mples were assayed after elution without further concentration. When larger volumes of water (up to 40 liters) were tested after elution and concentra tion with PEG, 38, 19, and 14% of the seeded Pan-1 were recovered from fini shed, ground, and surface waters, respectively. The limit of detection of P an-1 by RT-PCR was estimated to be 0.75 to 1.5 PFU in 40 liters of finished water. This method may be adapted for monitoring HuCVs in drinking water a nd other types of water for public health safety.