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

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• 作者：Vilaginè ; s ; Ph. ; Suarez ; A. ; Sarrette ; B. ; Vilaginè ; s ; R.
• 关键词：PEG concentration ; RT-PCR ; poliovirus ; rotavirus ; drinking water
• 刊名：Water Science and Technology
• 出版年：1997
• 出版时间：1997
• 年：1997
• 卷：35
• 期：11-12
• 页码：455-459
• 全文大小：266 K

文摘

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 Trizol^TM 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.