Crystal digital RT-PCR for the detection and quantification of norovirus and hepatitis A virus RNA in frozen raspberries
Crystal digital RT-PCR for the detection and quantification of norovirus and hepatitis A virus RNA in frozen raspberries
Simple item page
Full item details
- creativework.keywords - en
- Hépatite A
- Hepatitis A
- Frozen fruit
- creativework.keywords - fr
- Fruits congelés
- Noroviruses
- Norovirus
- dc.contributor.author
- Larocque, Émilie
- Lévesque, Valérie
- Lambert, Dominic
- dc.date.accepted
- 2022-08-18
- dc.date.accessioned
- 2023-12-01T14:54:56Z
- dc.date.available
- 2023-12-01T14:54:56Z
- dc.date.issued
- 2022-11-02
- dc.date.submitted
- 2022-02-07
- dc.description.abstract - en
- Berries are important vehicles for norovirus (NoV) and hepatitis A virus (HAV) foodborne outbreaks. Sensitive and quantitative detection of these viruses in food samples currently relies on RT-qPCR, but remains challenging due to their low concentration and the presence of RT-qPCR inhibitors. Moreover, quantification requires a standard curve. In this study, crystal digital RT-PCR (RT-cdPCR) assays were adapted from RT-qPCR sets of primers and probe currently used in our diagnostic laboratory for the detection and precise quantification of norovirus genogroups I and II (NoV GI, GII) and hepatitis A virus (HAV) RNA in frozen raspberry samples. We selected assay conditions based on optimal separation of positive and negative droplets, and peak resolution. Using virus-specific in vitro RNA transcripts diluted in raspberry RNA extracts, we showed that all three RT-cdPCR assays were sensitive, and we estimated the 95 % detection limit at 9 copies per RT-cdPCR reaction for NoV GI, 3 for NoV GII, and 14 for HAV. Serial dilutions of the RNA transcripts showed excellent linearity over a range of four orders of magnitude. We achieved precise quantification (CV ≤ 35 %) of the RNA transcripts between runs down to 15–145 copies per reaction for NoV GI, <20 for NoV GII, and < 15 for HAV. The three RT-cdPCR assays also proved to be tolerant to inhibitors from frozen raspberries, although not as tolerant as the RT-qPCR assays in the case of NoV GI and HAV. We further evaluated the assays with inoculated frozen raspberry samples and compared their performance to that of the RT-qPCR assays. As compared to the corresponding RT-qPCR assays, the NoV GI and HAV RT-cdPCR assays showed a decreased qualitative sensitivity, while the NoV GII RT-cdPCR assay had an increased sensitivity. As for quantification, the NoV GI and NoV GII RT-cdPCR assays produced similar estimates of RNA copy number than their respective RT-qPCR assays, whereas for HAV, the RT-cdPCR assay produced lower estimates than the RT-qPCR assay. However, all the RT-cdPCR assays provided more precise quantitative measurements at low levels of contamination than the RT-qPCR assays. In conclusion, the potential of the RT-cdPCR assays in this study to detect viral RNA from frozen raspberries varied according to assay, but these RT-cdPCR assays should be considered for precise absolute quantification in difficult matrices such as frozen raspberries.
- dc.identifier.citation
- Larocque, É., Lévesque, V., & Lambert, D. (2022). Crystal Digital RT-PCR for the detection and quantification of norovirus and hepatitis A virus RNA in Frozen Raspberries. International Journal of Food Microbiology, 380, 109884. https://doi.org/10.1016/j.ijfoodmicro.2022.109884
- dc.identifier.doi
- https://doi.org/10.1016/j.ijfoodmicro.2022.109884
- dc.identifier.uri
- https://open-science.canada.ca/handle/123456789/1334
- dc.language.iso
- en
- dc.publisher
- Elsevier
- dc.rights - en
- Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)
- dc.rights - fr
- Creative Commons Attribution - Pas d'utilisation commerciale - Pas de modification 4.0 International (CC BY-NC-ND 4.0)
- dc.rights.openaccesslevel - en
- Gold
- dc.rights.openaccesslevel - fr
- Or
- dc.rights.uri - en
- https://creativecommons.org/licenses/by-nc-nd/4.0/
- dc.rights.uri - fr
- https://creativecommons.org/licenses/by-nc-nd/4.0/deed.fr
- dc.subject - en
- Agriculture
- dc.subject - fr
- Agriculture
- dc.subject.en - en
- Agriculture
- dc.subject.fr - fr
- Agriculture
- dc.title - en
- Crystal digital RT-PCR for the detection and quantification of norovirus and hepatitis A virus RNA in frozen raspberries
- dc.type - en
- Article
- dc.type - fr
- Article
- local.acceptedmanuscript.articlenum
- 109884
- local.article.journaltitle
- International Journal of Food Microbiology
- local.article.journalvolume
- 380
- local.peerreview - en
- Yes
- local.peerreview - fr
- Oui
Download(s)
Original bundle
1 - 1 of 1
Name: Emilie Larocque - Crystal digital RT-PCR.PDF
Size: 2.71 MB
Format: PDF
Collection(s)