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 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.
Shieh
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Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill 27599, USA.
Wait
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Tai
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Sobsey
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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
D017931:DNA Primers; D004279:DNA, Viral; D006146:Guanidines; D017879:Isothiocyanates; D012367:RNA, Viral; D012722:Sewage; C054435:guanidine isothiocyanate