Norovirus Extraction from Frozen Raspberries Using Magnetic Silica Beads

Abstract

Human noroviruses (HuNoV) are among the main causes of acute gastroenteritis worldwide. Frozen raspberries have been linked to several HuNoV food-related outbreaks. However, the extraction of HuNoV RNA from frozen raspberries remains challenging. Recovery yields are low, and real-time quantitative reverse transcriptase PCR (RT-qPCR) inhibitors limit the sensitivity of the detection methodologies. A new approach using fine magnetic silica beads was developed for the extraction of HuNoV spiked on frozen raspberries. Relatively low recovery yields were observed with both the magnetic silica bead and the reference ISO 15216-1:2017 methods. High RT-qPCR inhibition was observed with the ISO 15216-1:2017 recommended amplification kit but could be reduced by using an alternative kit. Reducing RT-qPCR inhibition is important to limit the number of inconclusive HuNoV assays thus increasing the capacity to assess the HuNoV prevalence in frozen raspberries.

Detection and Sequencing of Multiple Human Norovirus Genotypes from Imported Frozen Raspberries Linked to Outbreaks in the Province of Quebec, Canada, in 2017

“…Frozen raspberries from bags labeled as whole individually quick frozen (IQF) collected at local stores were used to prepare 25 g subsamples in order to perform artificial contamination experiments. Aliquots of clarified 10% stool sample (HuNoV GII.4 CFIA-FVR-019) were prepared as described before (Raymond et al, 2021). They were vortexed 2 s and diluted in PBS to a final volume of 100 µl per subsample at the genomic equivalent copy (gEq) level needed.…”

“…The HID50 of the HuNoV Norwalk strain was estimated at 1320 (95% CI 440-3760) genomic equivalent (gEq) in serogroups O and A (Atmar et al, 2014). We reported previously that the MSB HuNoV GI and GII recovery yields range from 2.6 to 5.7% (Raymond et al, 2021). Based on the Poisson distribution (Fig.…”

“…Detection of HuNoV relies on viral recovery from food matrix, extraction of RNA and reverse transcription-polymerase chain reaction (RT-PCR) amplification methodologies (Vinje, 2015 ). The extraction of HuNoV from contaminated frozen raspberry matrices are associated with low recovery (2–9%) (Raymond et al, 2021 ; Summa & Maunula, 2018 ). Presence of PCR inhibitors is frequently reported in RNA extracts from soft fruits and can lead to false negative or inconclusive results (De Keuckelaere et al, 2015 ; Fraisse et al, 2017 ; Raymond et al, 2021 ; Summa & Maunula, 2018 ).…”

“…The extraction of HuNoV from contaminated frozen raspberry matrices are associated with low recovery (2–9%) (Raymond et al, 2021 ; Summa & Maunula, 2018 ). Presence of PCR inhibitors is frequently reported in RNA extracts from soft fruits and can lead to false negative or inconclusive results (De Keuckelaere et al, 2015 ; Fraisse et al, 2017 ; Raymond et al, 2021 ; Summa & Maunula, 2018 ). When the inhibition is above 75%, it leads to an “invalid result” according to ISO 15216-1:2017 (ISO, 2017 ).…”

“…A magnetic silica bead (MSB) approach for the extraction of norovirus was recently developed (Raymond et al, 2021 ). In this study, its application during HuNoV outbreak investigations linked to contaminated frozen raspberries in Canada, in 2017, is presented.…”