Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland.
J Appl Microbiol. 2018 Apr;124(4):1008-1016. doi: 10.1111/jam.13737. Epub 2018 Mar 12.
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.
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 log reductions in the titre of faecal rotavirus.
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.
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.
检测/定量 RNA 病毒主要通过逆转录酶（RT）-（q）PCR 进行，但它无法区分感染性和非感染性病毒。我们的目的是测试不同的 RT-qPCR 前处理方法，以消除源自外部核酸或受损颗粒基因组的阳性结果。
将热灭活（80°C 10 分钟）轮状病毒 Wa 株和含有轮状病毒或诺如病毒的粪便样本用 PMA/PMAxx、苯甲脒酶或粗提 RNA 酶进行预处理。尽管 PMA/PMAxx 预处理对 RV 并不总是有效，但它们似乎对热灭活的诺如病毒在一定程度上有效。苯甲脒酶和 RNA 酶可使粪便轮状病毒的滴度降低 2.2-2.8 个对数级。
所有预处理方法都需要针对每种病毒单独进一步验证，同时考虑样本基质和失活动力学条件。虽然没有一种预处理方法能使失活病毒完全无法检测到，但 RNA 酶对轮状病毒和诺如病毒都最有效。
本研究揭示了最常见的 RT-qPCR 前处理方法在热处理后消除受损、非感染性轮状病毒和诺如病毒的能力。据我们所知，这是苯甲脒酶首次在这种情况下被使用。
