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 contamination of water by viruses, including HuCVs. We developed a method to concentrate and detect HuCVs in water samples by using a cultivable primate calicivirus (Pan-1) 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 transcription (RT)-PCR following extraction of the RNA with Trizol. Pan-1 was more sensitive to high-pH treatment than poliovirus was; a pH 9.0 BE solution was found to recover 35% more viable Pan-1 than a pH 9.5 BE solution recovered. Pan-1 was recovered from small volumes of deionized, finished, ground, and surface waters at efficiencies of 94, 73, 67, and 64%, respectively, when samples were assayed after elution without further concentration. When larger volumes of water (up to 40 liters) were tested after elution and concentration with PEG, 38, 19, and 14% of the seeded Pan-1 were recovered from finished, ground, and surface waters, respectively. The limit of detection of Pan-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 and other types of water for public health safety.
Huang
P W
PW
Center for Pediatric Research, Children's Hospital of The King's Daughters and Eastern Virginia Medical School, Norfolk, Virginia 23510-1001, USA.
Laborde
D
D
Land
V R
VR
Matson
D O
DO
Smith
A W
AW
Jiang
X
X
D002139:Caliciviridae / Q000145:classification / Q000302:isolation & purification*; D017250:Caliciviridae Infections / Q000453:epidemiology / Q000635:transmission; D004196:Disease Outbreaks; D005759:Gastroenteritis / Q000821:virology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017955:Poliovirus / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014871:Water Microbiology
D012367:RNA, Viral