S Oristo1, H-J Lee1, L Maunula1. 1. Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland.
Abstract
AIMS: Detection/Quantification of RNA viruses is mostly done by reverse-transcriptase (RT)-(q)PCR, but it does not distinguish between infectious and noninfectious viruses. Our aim was to test, how different pretreatments before RT-qPCR could eliminate positivity originated from external nucleic acids or genomes of damaged particles. METHODS AND RESULTS: Heat-inactivated (80°C for 10 min) rotavirus Wa strain and faecal samples containing rotavirus or norovirus were treated with PMA/PMAxx, benzonase or crude extract RNase prior to RT-qPCR. PMA/PMAxx pretreatments were not consistently efficient for RV, although they seemed to work to some extent for heat-inactivated norovirus. Benzonase and RNase provided consistently 2·2-2·8 log10 reductions in the titre of faecal rotavirus. CONCLUSIONS: All pretreatments need to be further validated for each virus separately, taking into account sample matrix and inactivation conditions. Although none of the pretreatments could completely render inactivated viruses undetectable, RNase worked most consistently for both rota- and norovirus. SIGNIFICANCE AND IMPACT OF THE STUDY: This study sheds light on capacity of the most common pre-RT-qPCR treatments to eliminate damaged, noninfectious rotaviruses and noroviruses after thermal treatment. To our knowledge, this is the first time, when benzonase has been used in this context.
AIMS: Detection/Quantification of RNA viruses is mostly done by reverse-transcriptase (RT)-(q)PCR, but it does not distinguish between infectious and noninfectious viruses. Our aim was to test, how different pretreatments before RT-qPCR could eliminate positivity originated from external nucleic acids or genomes of damaged particles. METHODS AND RESULTS: Heat-inactivated (80°C for 10 min) rotavirus Wa strain and faecal samples containing rotavirus or norovirus were treated with PMA/PMAxx, benzonase or crude extract RNase prior to RT-qPCR. PMA/PMAxx pretreatments were not consistently efficient for RV, although they seemed to work to some extent for heat-inactivated norovirus. Benzonase and RNase provided consistently 2·2-2·8 log10 reductions in the titre of faecal rotavirus. CONCLUSIONS: All pretreatments need to be further validated for each virus separately, taking into account sample matrix and inactivation conditions. Although none of the pretreatments could completely render inactivated viruses undetectable, RNase worked most consistently for both rota- and norovirus. SIGNIFICANCE AND IMPACT OF THE STUDY: This study sheds light on capacity of the most common pre-RT-qPCR treatments to eliminate damaged, noninfectious rotaviruses and noroviruses after thermal treatment. To our knowledge, this is the first time, when benzonase has been used in this context.
Authors: Mats Leifels; Dan Cheng; Emanuele Sozzi; David C Shoults; Stefan Wuertz; Skorn Mongkolsuk; Kwanrawee Sirikanchana Journal: Water Res X Date: 2020-12-09