Enzymatic Pre-treatment of Wastewater to Minimize Recovery by Reverse Transcriptase PCR of RNA from Inactive Bacteriophages

Abstract

Quantitative viral risk assessments for wastewaters are notoriously difficult. The often considered quantitative reverse transcriptase PCR reflects poorly on virus infectivity rates leading to inaccurate risk interpretations. Various techniques focused on the degradation of the nucleic acids of non-infective viruses were previously employed. We comparatively assessed the effectiveness of such enzymatic treatments for MS2 bacteriophage in treated wastewaters. The single use of RNase A at an appropriate concentration may be as effective as the combination of RNase followed by Proteinase K and more rapid. While all tested enzymatic treatments minimized recovery of RNA (>95 %) in the absence of infective MS2, none completely eliminated the signal recovery. Selection of any enzymatic protocol for minimizing recovery of RNA from degraded, non-infective viruses should balance the methods efficacy with its expediency.

Virus Removal and Inactivation Mechanisms during Iron Electrocoagulation: Capsid and Genome Damages and Electro-Fenton Reactions

“…Viral RNA concentrations both in the bulk water and flocs were quantified using one-step real-time RT-qPCR (SI Section S8). , The chosen amplicon (77 bp) targets 2.2% of the genome and is widely employed because of its high sensitivity and specificity. ,− …”

Real-Time qPCR as a Method for Detection of Antibody-Neutralized Phage Particles

“…This method can also be applied in other experiments that require detection of a “neutralized” phage, which is merely a phage that is not active against bacteria. Notably, qPCR has already been applied for phage detection in other (than phage neutralizing) conditions: in phage cultures ( Edelman and Barletta, 2003 ; Clokie, 2009 ; Anderson et al, 2011 ; Refardt, 2012 ; Dieterle et al, 2016 ), food ( Imamovic and Muniesa, 2011 ; Flannery et al, 2014 ; Perrin et al, 2015 ; Parente et al, 2016 ; Hartard et al, 2017 ; Muhammed et al, 2017 ), environmental samples ( Farkas et al, 2015 ; Kunze et al, 2015 ; Unnithan et al, 2015 ; Mankiewicz-Boczek et al, 2016 ), and in feces ( Imamovic et al, 2010 ; Chehoud et al, 2016 ), where some interference from antibodies cannot be excluded ( Majewska et al, 2015 ).…”

“…Bacteriophages have been quantitatively analyzed and discriminated by real-time qPCR directly in microbiological cultures, and the authors found this method to be a good alternative to the plaque assay ( Edelman and Barletta, 2003 ; Clokie, 2009 ; Anderson et al, 2011 ; Refardt, 2012 ; Dieterle et al, 2016 ). Real-time PCR has been further demonstrated as applicable for a rapid screening allowing phage detection in food (milk, fruits, vegetables, seafood, meat) ( Imamovic and Muniesa, 2011 ; Flannery et al, 2014 ; Perrin et al, 2015 ; Parente et al, 2016 ; Hartard et al, 2017 ) and water samples ( Farkas et al, 2015 ; Kunze et al, 2015 ; Unnithan et al, 2015 ; Mankiewicz-Boczek et al, 2016 ) or in feces ( Imamovic et al, 2010 ; Chehoud et al, 2016 ). However, potential applicability of real-time qPCR for detection of inactivated (non-infective) but still biologically active (e.g., immunoreactive) phage has never been investigated.…”