ENZYMATIC AMPLIFICATION OF ENTERIC VIRUSES FROM WASTEWATERS

The challenge to use of enzymatic amplification for detection of waterborne viruses is the effective removal of a range of inhibitory components present in environmental samples. This study combines a number of individual processes to simultaneously concentrate and purify enteric viruses from wastewaters. The procedure of secondary concentration and purification, using chloroform extraction, polyethylene glycol precipitation, Sephadex G-200 gel filtration, and ultrafiltration, was shown to be 39 and 31 % efficient in recovering enterovirus from raw sewage and oxidation pond effluent respectively, achieving a 100-fold reduction of the sample volume from 10 mL to 100 mu L. The secondary concentrates were analysed by reverse transcription and polymerase chain reaction, and the sensitivity was shown to be between 0.02-0.2 plaque forming units of enterovirus. This allows this theoretical detection of approximately 1 PFU/L of wastewater. The Tth polymerase was shown to be more effective for the amplification of virus in wastewaters than was Tag polymerase. Overall, the procedure enabled the sensitive detection of rotavirus, hepatitis A, and enterovirus from wastewaters.