The development of a dot blot assay using gene probes for the detection of enteroviruses in water

Abstract

Enteric viruses are viruses which replicate in the intestinal tract of man and animals. One mode of transmission for enteric viruses is the fecal-oral route. Drinking water which has been contaminated with sewage or sewage effluent has been implicated as a means for the spread of enteric viruses. Monitoring water for virus contamination requires two steps: 1) the collection and the concentration of the water sample and 2) the isolation and identification of the virus present. Current methods for the detection of enteric viruses in water requires the use of animal cell culture. This technique has several drawbacks, such as: 1) long incubation periods, up to two and three weeks, before some enteric viruses are detected, 2) not all viruses can be detected in one cell line, and 3) not all viruses have been grown in cell culture. More rapid techniques, such as fluorescent antibody or radioimmunoassay do not have the needed sensitivity to detect the low levels of virus found in contaminated water. These techniques also require the production of an antibody for each different virus type. An alternative technique for the detection of viruses in water was sought. Recent advances in recombinant DNA technology now makes it possible to detect viruses without the use of cell culture or antibodies. Gene probes that hybridize to the RNA of poliovirus and hepatitis A virus were tested for their ability to detect different enteric viruses. The probes were labeled with ³²P dCTP and ³²P dATP to a specific activity greater then 1.0 x 10⁹ cpm/ug DNA. Gene Screen Plus (NEN) was chosen as the hybridization membrane since it was more sensitive to virus detection than the other membranes tested. A dot-blot apparatus (Bio Rad) was used to apply the samples. Results were visualized by autoradiography for thirty-six hours at -70° C. One infectious unit of poliovirus and hepatitis A virus was detected using labeled cDNA probes. Upon comparison, the dot blot assay was as sensitive as tissue culture for the detection of poliovirus in beef extract, secondary effluent, and tapwater. Environmental samples, such as secondary effluent, reclaimed wastewater and unchlorinated drinking water were also assayed for poliovirus and hepatitis A virus with the use of gene probes. The results presented here offer an alternative method for screening water samples for the presence of enteric viruses.