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 vir al 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 biotinyl ated oligonucleotide probe was hybridized to poliovirus-RNA in solutio n. Streptavidin coated magnetic beads were then added to isolate the R NA-oligonucleotide hybrid. The procedure allows for the recovery of vi ral RNA suitable for amplification by reverse transcription-polymerase chain reaction (RT-PCR). This nucleic acid capture system was effecti ve in both concentrating, and purifying poliovirus RNA while removing environmental RT-PCR inhibitors. A detection sensitivity of one plaque forming unit (PFU) in 250 mu l 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 ac id capture RT-PCR was significantly greater than direct RT-PCR, when a pplied to environmental samples. The amplified product was sequenced t o ensure specificity. Furthermore, this technique is rapid, reliable a nd can be readily adapted to detect other viral pathogens. Copyright ( C) 1997 Elsevier Science B.V.