A METHOD TO REMOVE ENVIRONMENTAL INHIBITORS PRIOR TO THE DETECTION OFWATERBORNE ENTERIC VIRUSES BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION

A method was developed to remove environmental inhibitors from sample concentrates prior to detection of human enteric viruses using the rev erse transcription-polymerase chain reaction (RT-PCR). Environmental i nhibitors, concentrated along with viruses during water sample process ing, are removed by the method through a series of steps that includes dialysis, solvent extraction, ultrafiltration and glass purification. The method was tested by spiking sodium phosphate with poliovirus typ e 1 with or without humic or fulvic acids and then measuring virus rec overy by plaque assay and RT-PCR. Results of the study indicated that (i) 90% of the spiked virus could be recovered from samples at the end of the ultrafiltration step, (ii) virus was detected in the final elu ate of samples containing as much as 0.5 mg of humic acid or 5.0 mg of fulvic acid, and (iii) as little as 0.06 plaque forming units (PFU) w as detectable per RT-PCR reaction. These results indicate that the des cribed purification method along with RT-PCR is a feasible approach fo r detecting waterborne human enteric viruses in the presence of interf ering substances. Copyright (C) 1997 Elsevier Science B.V.