Reverse transcription PCR to detect enteroviruses in surface water

Abstract

We have developed a simple, fast, and efficient procedure to detect enteroviruses in water samples. Aliquots of water are subjected to two-step filtration, with the second filter containing a positively charged nylon membrane that holds back virus particles. Viruses thus adsorbed are directly lysed, and RNA is isolated by hybridization to specific oligonucleotides bound to magnetic beads. The solution used contains guanidine thiocyanate, which lyses virus particles, inactivates enzymes, e.g., RNases, allows mild hybridization conditions, and does not influence biotin-streptavidin interaction on magnetic beads. Detection and specific identification are accomplished by reverse transcription PCR of the highly conserved noncoding region at the 5' end of virus RNA combined with Southern hybridization. The system was tested with tap water artificially spiked with poliovirus vaccine and yielded a detection limit of 20 50% tissue culture infective doses per liter. We used the same procedure to investigate the water quality of surface water at public beaches by rivers and lakes. Of 40 samples tested, 7 were positive for enteroviruses. A comparison with enterobacterial contamination determined by PCR and classical microbiological methods in parallel showed that enteroviruses were found only in samples also positive for Escherichia coli. In conclusion, this procedure can easily be adapted to test large water samples and is simple enough to be used for routine determinations of water quality in terms of virus contamination.