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Performance of pre‐ RT ‐ qPCR treatments to discriminate infectious human rotaviruses and noroviruses from heat‐inactivated viruses: applications of PMA / PMA xx, benzonase and RN ase
Author(s) -
Oristo S.,
Lee H.J.,
Maunula L.
Publication year - 2018
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.13737
Subject(s) - virology , biology , microbiology and biotechnology , norovirus , rnase p , virus , rna , gene , genetics
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 / PMA xx, benzonase or crude extract RN ase prior to RT ‐ qPCR . PMA / PMA xx pretreatments were not consistently efficient for RV , although they seemed to work to some extent for heat‐inactivated norovirus. Benzonase and RN ase provided consistently 2·2–2·8 log 10 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, RN ase 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.