Development of a nucleic acid capture probe with reverse transcriptase-polymerase chain reaction to detect poliovirus in groundwater.
There is a need to develop a practical method for the detection of viral contaminates in water supplies. In this study, poliovirus was used as a model to develop a nucleic acid capture technique. This technique was used to recover viral RNA from concentrated groundwater samples. Poliovirus RNA was isolated using magnetic bead technology. A biotinylated oligonucleotide probe was hybridized to poliovirus-RNA in solution. Streptavidin coated magnetic beads were then added to isolate the RNA-oligonucleotide hybrid. The procedure allows for the recovery of viral RNA suitable for amplification by reverse transcription-polymerase chain reaction (RT-PCR). This nucleic acid capture system was effective in both concentrating, and purifying poliovirus RNA while removing environmental RT-PCR inhibitors. A detection sensitivity of one plaque forming unit (PFU) in 250 microliters of a concentrated environmental sample was routinely attained. This was the same detection level found with seeded purified water. It was shown that the sensitivity of nucleic acid capture RT-PCR was significantly greater than direct RT-PCR, when applied to environmental samples. The amplified product was sequenced to ensure specificity. Furthermore, this technique is rapid, reliable and can be readily adapted to detect other viral pathogens.
Regan
P M
PM
Winchester Engineering and Analytical Center, U.S. Food and Drug Administration, MA 01890, USA.
Margolin
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D004279:DNA, Viral / Q000032:analysis; D005618:Fresh Water / Q000821:virology*; D006801:Humans; D008280:Magnetics; D017955:Poliovirus / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis; D012194:RNA-Directed DNA Polymerase
D004279:DNA, Viral; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase