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To examine the effect of chlorine on viral RNA and infectivity, chlorine demand-freebuffer was spiked with ~10,000 pfu/ml of poliovirus 2, echovirus 7 or coxsackie B5. Thesamples were then subjected to chlorine disinfection over 30 minutes with samples takenat various time periods with a 10 ml aliquot taken from each time point for analysis byreal-time RT-PCR and cell culture. A 1 mL sample was then extracted using theEpicentre Masterpure total nucleic acid extraction kit and analyzed by real-time RT-PCR.For the real-time RT-PCR reaction, 5 ml of sample were amplified using ABI AgPathone-step RT-PCR mastermix using primers and probes. Extractions were also examined by a real-time PCR inhibition assay to determine if the extractions contained inhibitors. The inhibition assay used 5 ml of sample added to a TaqMan one-step mastermix containing a control DNA sample, primers and probesprovided in the ABI exogenous internal control kit. Five positive control samples containing the inhibitor contained in the kit and five negative controls containing just water were also included. All real-time assay were run on ABI FAST 96-well optical plates. Includes extended abstract only.

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Edition: Vol. - No. Published: 11/01/2009 Number of Pages: 2File Size: 1 file , 720 KB