Development of a nucleic acid capture probe with reverse transcriptase-polymerase chain reaction to detect poliovirus...

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.