METHODS TO REMOVE INHIBITORS IN SEWAGE AND OTHER FECAL WASTES FOR ENTEROVIRUS DETECTION BY THE POLYMERASE CHAIN-REACTION

Typical environmental sample concentration procedures developed to purify virions are not always compatible with reverse transcription-polymerase chain reaction (RT-PCR). The processing steps of polyethylene glycol (PEG) precipitation and ultrafiltration were found to enrich not only virions but also certain RT-PCR inhibitors. Inhibitors were eliminated by a single-step guanidinium isothiocyanate (GIT) extraction to purify and precipitate viral genomic RNAs immediately prior to RT-PCR. The detection sensitivity of GIT extraction-RT-PCR was found to be 0.6-0.003 50% tissue culture infectious doses (TCID50) for 9 enteroviruses in infected cell extracts. When sewage, concentrated up to 385,000-fold and seeded with 1-3 plaque-forming units of poliovirus, was extracted with GIT solution, viruses were detectable in samples originally judged negative by direct RT-PCR without GIT extraction. Eleven waste samples (3 sewage, 5 latrine solids, 2 gauze pad extracts and 1 stool) processed by a series of steps that included PEG precipitation, solvent extractions, and Sephadex G-200 chromatography were examined for enteroviruses by RT-PCR, both with and without GIT extraction. GIT extraction eliminated sample inhibitory substances and increased the proportion of enterovirus positive samples from 3 to 7 of 11. GIT extraction in conjunction with virion concentration improves RT-PCR detection of viruses in sewage and other fecal wastes.