title: Methods to remove inhibitors in sewage and other fecal wastes for enterovirus detection by the polymerase chain reaction.
abstract: 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 pads 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.
authors.affiliation: Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill 27599, USA.
authors.forename: Y S
authors.initials: YS
authors.lastname: Shieh
authors.forename: D
authors.initials: D
authors.lastname: Wait
authors.forename: L
authors.initials: L
authors.lastname: Tai
authors.forename: M D
authors.initials: MD
authors.lastname: Sobsey
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D004770:Enterovirus / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology*; D006146:Guanidines; D006801:Humans; D017879:Isothiocyanates; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis; D012680:Sensitivity and Specificity; D012722:Sewage / Q000821:virology*; D014158:Transcription, Genetic; D014481:United States
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D006146:Guanidines; D017879:Isothiocyanates; D012367:RNA, Viral; D012722:Sewage; C054435:guanidine isothiocyanate