A31 Molecular characterization of circulating human noroviruses in Canada to assess RT-qPCR assays used for the detection of foodborne noroviruses
Bibliographic record
Abstract
Noroviruses (NoV) have been identified as the main cause of acute gastroenteritis in humans of all ages and as the main etiologic agent of foodborne illnesses worldwide. Since human NoV are difficult to grow in vitro, and that alternative diagnostic methods such as electron microscopy and serology lack sensitivity, modern detection methods rely on molecular approaches such as Reverse Transcription qPCR (RT-qPCR). However, the continuous emergence of new NoV strains, coupled with the genetic diversity between and within genotypes and the potential for recombination, represent continuous and significant challenges to clinical and food diagnostic laboratories. In this study, we investigated 215 outbreak-related NoV isolates collected between 2010 and 2016 by the Quebec provincial public health laboratory (Laboratoire de santé publique du Québec). An ∼2.4-kb-long amplicon, encompassing the 3′-end of ORF1, polymerase gene, and the complete VP1 gene, was characterized using Next-Generation Sequencing. Paired-end dual-indexed sequencing runs of 2×75 cycles were conducted on pooled libraries on Illumina’s MiSeq Instrument and paired reads were assembled de novo into a single contig. Firstly, both the 3′-end of ORF1 and VP1 sequences derived from the contig were genotyped and subtyped by phylogenetic analysis. Typing revealed that multiple NoV genotypes were in circulation between 2010 and 2016, but NoV genotype GII.4 was responsible for most of the gastroenteritis outbreaks. Moreover, genotyping of ORF1 and VP1 did not always match suggesting recombinant noroviruses. Secondly, binding affinity between all the characterized NoV isolates and the primers or probes was assessed in silico. The analysis demonstrated that mismatches in the Taqman probe-binding sites were associated with certain genotypes: GI.P3_GI.3a, GI.P9_GI.9, GI.Pa_GI.3 b, GII.P7_GII.6, and GII.P17_GII.17. The NoV GII Taqman probe was modified accordingly and tested against the original probe using a panel of twelve representative samples. A single mismatch in the NoV GII Taqman probe impeded detection: concurrent testing demonstrated a discrepancy between the CT values. Improved understanding of the genetic diversity of circulating noroviruses is important to develop molecular assays with appropriate inclusivity and exclusivity panels during analytical sensitivity evaluations in food virology laboratories.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.002 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.002 | 0.003 |
| Science and technology studies | 0.003 | 0.001 |
| Scholarly communication | 0.002 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.003 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".