Comparison and optimization of detection methods for noroviruses in frozen strawberries containing different amounts of RT-PCR inhibitors

Abstract

Frozen berries have been repeatedly identified as vehicles for norovirus (NoV) transmission causing large gastroenteritis outbreaks. However, virus detection in berries is often hampered by the presence of RT-PCR-inhibiting substances. Here, several virus extraction methods for subsequent real-time RT-PCR-based NoV-RNA detection in strawberries were compared and optimized. NoV recovery rates (RRs) between 0.21 ± 0.13% and 10.29 ± 6.03% were found when five different artificially contaminated strawberry batches were analyzed by the ISO/TS15216-2 method indicating the presence of different amounts of RT-PCR inhibitors. A comparison of five different virus extraction methods using artificially contaminated strawberries containing high amounts of RT-PCR inhibitors revealed the best NoV RRs for the ISO/TS15216 method. Further improvement of NoV RRs from 2.83 ± 2.92% to 15.28 ± 9.73% was achieved by the additional use of Sephacryl(®)-based columns for RNA purification. Testing of 22 frozen strawberry samples from a batch involved in a gastroenteritis outbreak resulted in 5 vs. 13 NoV GI-positive and in 9 vs. 20 NoV GII-positive samples using the original ISO/TS15216 method vs. the extended protocol, respectively. It can be concluded that the inclusion of an additional RNA purification step can increase NoV detection by the ISO/TS15216-2 method in frozen berries containing high amounts of RT-PCR inhibitors.

Application of an Optimized Direct Lysis Method for Viral RNA Extraction Linking Contaminated Dates to Infection With Hepatitis A Virus

“…However, since extractions of the positive dates were not conducted using the modified ISO 15216-1 method (ISO, 2017), we cannot exclude that this could also have resulted in a similar outcome. This widely used EU standard method for detection of viruses on soft fruit can be inefficient in recovering RT-qPCR detectable viral RNA from different types of soft fruits (Bartsch et al, 2016;Rajiuddin et al, 2020) including dates (Boxman et al, 2012). The reason is that food matrices may challenge efficient viral elution from the matrix surfaces and contain organic and inorganic substances that interfere with RT-qPCR detection of target sequences (Schrader et al, 2012;Perrin et al, 2015).…”

“…Previous studies using alternative variations of a direct lysis methods for extraction of spiked NoV (Bartsch et al, 2016) or model viruses have reported challenges with RT-qPCR inhibition, which could be overcome by dilution or filtration of extracts before RT-qPCR detection (Sun et al, 2019). In our study an inhibition >75% was observed for only NoV GI RT-qPCR detection in four sub-extractions from the dates, which thus failed the quality criteria described in the ISO 15216-1 (ISO, 2017), although extracts from the remaining subsamples and a 10-fold dilution of the inhibited subsamples did comply with this criteria.…”

“…In addition, the ISO standard is only validated for detection of NoV and HAV in the matrices described (soft fruit, leafy greens, stem and bulb vegetables, bottled water, mussels and oysters), excluding other types of potential vehicles of virus transmission, which limits the scope of the standard. Perrin et al (2015) proposed a direct viral lysis method from soft-fruit by immersion in lysis buffer, which enabled efficient detection of NoV surrogates on spiked raspberries, although it was less efficient in analyzing strawberries for the detection of spiked NoV (Bartsch et al, 2016). A slightly modified version in which inhibition could be overcome by filtering the RNA extract before molecular detection, confirmed the applicability of the method principle on samples of different berry types spiked with model viruses (Sun et al, 2019).…”

Hepatitis A outbreak linked to imported frozen strawberries by sequencing, Sweden and Austria, June to September 2018

“…Moreover, the viral load in berries is often low and the viral particles have to be concentrated before performing RT real-time PCR. Many concentration methods are inefficient and may cause co-purification of RT and/or PCR inhibitors present in the food sample [ 19 ]. These factors in combination can lead to an underestimation of the viral load as described for other enteric viruses [ 20 ]; false negative results are therefore common.…”

Simultaneous Detection of Four Foodborne Viruses in Food Samples Using a One-Step Multiplex Reverse Transcription PCR

“…In the case of oysters, it is known that the midgut can specifically bind virus particles [11]. Fresh lettuce and frozen berries are problematic because they can harbor HAV [4,[28][29][30]. In Korea, a less fermented vegetable product is infrequently associated with acute gastroenteritis in humans, likely due to the use of untreated groundwater [9].…”