Enzyme Treatment RT - PCR to Differentiate Infectious and Inactivated F + RNA 1 Phages

F+RNA phages are recommended as indicators of fecal contamination, the presence 5 of enteric viruses, and as viral surrogates to elucidate the resistance of viruses to adverse 6 conditions or assess the effectiveness of inactivating processes. RT-PCR methods have been used 7 to detect, quantify or identify subgroups of F+RNA phages. However, these methods may 8 overestimate the infectivity of F+RNA phages in test samples since the presence of both 9 infectious and inactivated phages (or naked RNA) can lead to positive RT-PCR signals. In this 10 study, we evaluated the ability of an enzyme treatment (ET) with proteinase K and RNase A prior 11 to RNA extraction followed by RT-PCR to differentiate infectious and inactivated F+RNA 12 phages. Results indicated that ET RT-PCR reduced, but not completely eliminated, false-positive 13 signals encountered by RT-PCR alone. The two-step ET RT-PCR, in which the enzymes were 14 added sequentially, was more effective at reducing false-positive signals than the one-step ET 15 RT-PCR, which involved addition of both enzymes together. Despite its inability to completely 16 eliminate false-positive signals, ET RT-PCR gave more reliable information on the infectivity of 17 F+RNA phages. Thus, this method is better than RT-PCR alone to detect F+RNA phages as 18 indicators to assess the risk of fecal contamination by enteric pathogens, or to evaluate the 19 effectiveness of virus inactivating processes. 20 21 1 CRIFS, Department of Food Science, University of Guelph, 43McGilvray St, Guelph, ON, 22 Canada, N1G2W1 23 * Corresponding author. CRIFS, Department of Food Science, University of Guelph, 24 43McGilvray St, Guelph, ON, Canada, N1G2W1; Email: [email protected]; Phone Number: 25 519-824-4120 x52269 26 AEM Accepts, published online ahead of print on 21 March 2014 Appl. Environ. Microbiol. doi:10.1128/AEM.03964-13 Copyright © 2014, American Society for Microbiology. All Rights Reserved. on O cber 6, 2017 by gest ht://aem .sm .rg/ D ow nladed fom