An improved method for concentrating rotavirus from water samples.

A modified adsorption-elution method for the concentration of seeded rotavirus from water samples was used to determine various factors which affected the virus recovery. An enzyme-linked immunosorbent assay was used to detect the rotavirus antigen after concentration. Of the various eluents compared, 0.05M glycine, pH 11.5 gave the highest rotavirus antigen recovery using negatively charged membrane filtration whereas 2.9% tryptose phosphate broth containing 6% glycine; pH 9.0 was found to give the greatest elution efficiency when a positively charged membrane was used. Reconcentration of water samples by a speedVac concentrator showed significantly higher rotavirus recovery than polyethylene glycol precipitation through both negatively and positively charged filters (p-value <0.001). In addition, speedVac concentration using negatively charged filtration resulted in greater rotavirus recovery than that using positively charged filtration (p-value = 0.004). Thirty eight environmental water samples were collected from river, domestic sewage, canals receiving raw sewage drains, and tap water collected in containers for domestic use, all from congested areas of Bangkok. In addition, several samples of commercial drinking water were analyzed. All samples were concentrated and examined for rotavirus antigen. Coliforms and fecal coliforms (0->1,800 MPN/100 ml) were observed but rotavirus was not detected in any sample. This study suggests that the speedVac reconcentration method gives the most efficient rotavirus recovery from water samples.

Detection of poliovirus, hepatitis A virus and rotavirus from sewage and water samples.

A modified adsorption-elution technique for concentration of enteric viruses from sewage and water samples was developed. The viruses in water were concentrated by negatively charged membrane filtration, eluted with 2.9% tryptose phosphate broth containing 6% glycine pH 9.0, and reconcentrated using centrifugation by a speedVac concentrator. The presence of poliovirus, hepatitis A virus (HAV) RNA, and rotavirus antigen was determined by cell culture isolation, nested polymerase chain reaction (nested PCR), and enzyme-linked immunosorbent assay (ELISA), respectively. A total of 100 sewage and water samples were collected from various sources in congested communities in Bangkok, concentrated and examined for those enteric viruses. Of 20 surface water samples from canals which located near sewage drains, 15% were positive for HAV RNA by nested PCR. Of 48 domestic sewage samples from man-holes of underground sewers, 8% were positive for rotavirus antigen by ELISA. Even though the samples were concentrated 256-2,000 fold, poliovirus was not found by isolation in cell culture.

Rapid detection of polioviruses in environmental water samples by one-step duplex RT-PCR.

This study describes the rapid detection of polioviruses in environmental waters by a simple reverse transcriptase-polymerase chain reaction (RT-PCR) using two primer pairs for differentiation of poliovirus from non-polio enteroviruses in a single reaction by a one-step method, combining RT and PCR in a single tube. The detection by agarose gel electrophoresis yielded 2 bands of 153-bp and 293-bp for poliovirus tested without the need for further hybridization. The detection sensitivity of this one-step duplex RT-PCR, as measured with RNA extracted by heat treatment from supernatant of infected cell extracts, was 10(-1) 50% tissue culture effective doses (TCID50). This assay was used to evaluate the ability of sample concentration by membrane filter-based adsorption and elution, and purification by a simple RNA isolation based on guanidine isothiocyanate-phenol-chloroform extraction; the system yielded a detection limit of 5 x 10(-1) TCID50 seeded in 5 liters of tap water. This protocol was applied to the poliovirus detection in environmental water collected from 2 communities in Bangkok, Thailand during February and May 1998. Of 100 samples tested, 2 water samples collected from the same open sewage pipeline at one location were positive for polioviruses and one sample collected from another sewage pipeline was positive for non-polio enterovirus while a further 97 water samples were negative for both polioviruses and non-polio enteroviruses. With poliovirus detection by cell culture technique, none of the 100 samples tested was positive for poliovirus type 1, 2 or 3. RT-PCR was more sensitive, rapid, simple and cost-effective than the cell culture technique since the two water samples which were positive for polioviruses by RT-PCR failed to be detected by cell culture. Sequence data of 293-bp amplicons from positive samples were compared with those of reference poliovirus strains in the Genbank and the EMBL databases and identity to the sequence of type 1 strain Sabin was found to be 99%.