Optimisation of the PEG reconcentration procedure for virus detection by cell culture or genomic amplification

A double reconcentration procedure was developed for virus detection in tapwater concentrates obtained by conventional adsorption-elution techniques suitable for cell inoculation as well as for genomic amplification. Using 7.5% PEG 6000 and 2.5% NaCl, a 15min contact time under agitation at room temperature followed by centrifugation (first step: 3,500xg, 90min, 4°C; second step 10,000xg, 20min, 4°C) were the conditions to obtain overall average virus recovery efficiencies of 71% for poliovirus from 900ml eluates and 88, 83 and 75% for poliovirus, coxsackie B2 and rotavirus respectively (400ml eluates). Direct extraction of viral RNA from the first PEG pellet with TrizolTM was efficient for RT-PCR assays without any further treatment. Primer pairs were selected to amplify rotavirus group A and poliovirus in seeded tapwater concentrated by adsorption elution through glass wool. A positive signal was obtained for theoretic virus concentration of 1 PFU. Analysis of field samples (1001) by cell culture and genomic amplification resulted in a higher sensitivity with the latter.