Nathalie Corneau

Development of a DNA Microarray for the Simultaneous Detection and Genotyping of Noroviruses

Current methods for detecting and genotyping noroviruses focus on the use of reverse transcriptase (RT)-mediated PCR. A major drawback of this approach is that short target RT-PCR products do not always encompass sequences that can be compared among research laboratories, resulting in difficulties for molecular epidemiology. We describe the use of a microarray-based system for simultaneous detection and molecular characterization of noroviruses. The protocol generates a 917-bp RT-PCR product that encompasses two major regions currently used for detection and analysis of norovirus genomes. The PCR products are then hybridized to an oligonucleotide array (NoroChip) based on 50-mer features, which allows for both confirmation of reaction specificity and molecular characterization of the amplified genome. Parallel sequence analyses of amplicons revealed that our microarray data were robust in separating genogroups I and II, and further subtyping to the cluster level was possible. This approach, combining detection and characterization, overcomes the need for expensive and time-consuming sequence analysis of amplified genome targets for molecular epidemiology.

Listeria monocytogenes infections

1. Historical aspects and contemporary problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .313 2. Characteristics of Listeria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .314 3. Nature of infection in man and animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .320 4. Prevalence of Listeria in foods, feeds and water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .320 5. Foods most often associated with human infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .327 6. Principles of detection of Listeria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .328 7. Treatment and prevention of listeriosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .330 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .331

Microfluidic Integration of a Cloth-Based Hybridization Array System (CHAS) for Rapid, Colorimetric Detection of Enterohemorrhagic Escherichia coli (EHEC) Using an Articulated, Centrifugal Platform.

We describe the translation of a cloth-based hybridization array system (CHAS), a colorimetric DNA detection method that is used by food inspection laboratories for colony screening of pathogenic agents, onto a microfluidic chip format. We also introduce an articulated centrifugal platform with a novel fluid manipulation concept based on changes in the orientation of the chip with respect to the centrifugal force field to time the passage of multiple components required for the process. The platform features two movable and motorized carriers that can be reoriented on demand between 0 and 360° during stage rotation. Articulation of the chip can be used to trigger on-the-fly fluid dispensing through independently addressable siphon structures or to relocate solutions against the centrifugal force field, making them newly accessible for downstream transfer. With the microfluidic CHAS, we achieved significant reduction in the size of the cloth substrate as well as the volume of reagents and wash solutions. Both the chip design and the operational protocol were optimized to perform the entire process in a reliable, fully automated fashion. A demonstration with PCR-amplified genomic DNA confirms on-chip detection and identification of Escherichia coli O157:H7 from colony isolates in a colorimetric multiplex assay using rfbO157, fliCH7, vt1, and vt2 genes.

Development and validation of a microarray for the confirmation and typing of norovirus RT-PCR products

Noroviruses are implicated in many worldwide institutional, food and waterborne outbreaks each year. Genetic typing of isolates is valuable for monitoring outbreak spread as well as variation in circulating strains. Microarrays have the potential to provide rapid genotype information for norovirus samples. The NoroChip v3.0 provides an oligonucleotide hybridization platform to screen for over 600 potential interactions in each experiment. The NoroChip v3.0 was developed at Health Canada and validated in seven international partner laboratories. Each laboratory validated the NoroChip v3.0 using norovirus amplicons routinely characterized in their testing protocols. Fragments from the capsid region (region C) and a 2.4 kb amplicon spanning polymerase and capsid sequences (region AD) were validated in six of the partner laboratories and provided correct genogroup typing information (GI or GII) when hybridized to the NoroChip v3.0. Results indicate that the current limiting factor for implementing the NoroChip v3.0 as a strain typing tool is the difficulty obtaining a long, specific amplicon from all circulating norovirus strains. Data obtained with the longer region AD amplicon provided the best discrimination between norovirus strains.

Prevalence and Molecular Characterization of the Hepatitis E Virus in Retail Pork Products Marketed in Canada

Infection with the hepatitis E virus (HEV) is very common worldwide. HEV causes acute viral hepatitis with approximately 20 million cases per year. While HEV genotypes 1 and 2 cause large waterborne and foodborne outbreaks with a significant mortality in developing countries, genotypes 3 and 4 are more prevalent in developed countries with transmission being mostly zoonotic. In North America and Europe, HEV has been increasingly detected in swine, and exposure to pigs and pork products is considered to be the primary source of infection. Therefore we set out to investigate the prevalence of HEV in retail pork products available in Canada, by screening meal-size portions of pork pâtés, raw pork sausages, and raw pork livers. The presence of the HEV genomes was determined by RT-PCR and viral RNA was quantified by digital droplet PCR. Overall, HEV was detected in 47% of the sampled pork pâtés and 10.5% of the sampled raw pork livers, but not in the sampled pork sausages, and sequencing confirmed that all HEV strains belonged to genotype 3. Further phylogenetic analysis revealed that except for one isolate that clusters with subtype 3d, all isolates belong to subtype 3a. Amino acid variations between the isolates were also observed in the sequenced capsid region. In conclusion, the prevalence of HEV in pâtés and raw pork livers observed in this study is in agreement with the current HEV distribution in pork products reported in other developed countries.

Enhancing the Detection of Giardia duodenalis Cysts in Foods by Inertial Microfluidic Separation

ABSTRACT The sensitivity and specificity of current Giardia cyst detection methods for foods are largely determined by the effectiveness of the elution, separation, and concentration methods used. The aim of these methods is to produce a final suspension with an adequate concentration of Giardia cysts for detection and a low concentration of interfering food debris. In the present study, a microfluidic device, which makes use of inertial separation, was designed and fabricated for the separation of Giardia cysts. A cyclical pumping platform and protocol was developed to concentrate 10-ml suspensions down to less than 1 ml. Tests involving Giardia duodenalis cysts and 1.90-μm microbeads in pure suspensions demonstrated the specificity of the microfluidic chip for cysts over smaller nonspecific particles. As the suspension cycled through the chip, a large number of beads were removed (70%) and the majority of the cysts were concentrated (82%). Subsequently, the microfluidic inertial separation chip was integrated into a method for the detection of G. duodenalis cysts from lettuce samples. The method greatly reduced the concentration of background debris in the final suspensions (10-fold reduction) in comparison to that obtained by a conventional method. The method also recovered an average of 68.4% of cysts from 25-g lettuce samples and had a limit of detection (LOD) of 38 cysts. While the recovery of cysts by inertial separation was slightly lower, and the LOD slightly higher, than with the conventional method, the sample analysis time was greatly reduced, as there were far fewer background food particles interfering with the detection of cysts by immunofluorescence microscopy.

Characterization of the Genomic Diversity of Norovirus in Linked Patients Using a Metagenomic Deep Sequencing Approach.

Norovirus (NoV) is the leading cause of gastroenteritis worldwide. A robust cell culture system does not exist for NoV and therefore detailed characterization of outbreak and sporadic strains relies on molecular techniques. In this study, we employed a metagenomic approach that uses non-specific amplification followed by next-generation sequencing to whole genome sequence NoV genomes directly from clinical samples obtained from 8 linked patients. Enough sequencing depth was obtained for each sample to use a de novo assembly of near-complete genome sequences. The resultant consensus sequences were then used to identify inter-host nucleotide variations that occur after direct transmission, analyze amino acid variations in the major capsid protein, and provide evidence of recombination events. The analysis of intra-host quasispecies diversity was possible due to high coverage-depth. We also observed a linear relationship between NoV viral load in the clinical sample and the number of sequence reads that could be attributed to NoV. The method demonstrated here has the potential for future use in whole genome sequence analyses of other RNA viruses isolated from clinical, environmental, and food specimens.

Rapid Screening and Identification of Living Pathogenic Organisms via Optimized Bioorthogonal Non-canonical Amino Acid Tagging

Pathogenic bacteria can be a major cause of illness from environmental sources as well as the consumption of contaminated products, giving rise to public health concerns globally. The surveillance of such living organisms in food and water supplies remains an important challenge in mitigating their deleterious societal effects. Here, we have developed an optimized bioorthogonal non-canonical amino acid tagging approach to the imaging, capture, and interrogation of shigatoxigenic/verotoxigenic Escherichia coli (VTEC) and Listeria that enables the distinction between living wild-type pathogenic bacteria. The approaches utilize homopropargylglycine (HPG), as well as optimized growth media, that restricts endogenous methionine biosynthesis in a variety of species of public health concern. Endogenous methionine residues are then replaced with HPG, which can then be modified using a myriad of compatible bioorthogonal reactions for tagging of exclusively live bacteria. The methods reported allow for the very rapid screening and identification of living pathogenic organisms.

Genetic characterization of norovirus GII.4 variants circulating in Canada using a metagenomic technique

BackgroundHuman norovirus is the leading cause of viral gastroenteritis globally, and the GII.4 has been the most predominant genotype for decades. This genotype has numerous variants that have caused repeated epidemics worldwide. However, the molecular evolutionary signatures among the GII.4 variants have not been elucidated throughout the viral genome.MethodA metagenomic, next-generation sequencing method, based on Illumina RNA-Seq, was applied to determine norovirus sequences from clinical samples.ResultsHerein, the obtained deep-sequencing data was employed to analyze full-genomic sequences from GII.4 variants prevailing in Canada from 2012 to 2016. Phylogenetic analysis demonstrated that the majority of these sequences belong to New Orleans 2009 and Sydney 2012 strains, and a recombinant sequence was also identified. Genome-wide similarity analyses implied that while the capsid gene is highly diverse among the isolates, the viral protease and polymerase genes remain relatively conserved. Numerous amino acid substitutions were observed at each putative antigenic epitope of the VP1 protein, whereas few amino acid changes were identified in the polymerase protein. Co-infection with other enteric RNA viruses was investigated and the astrovirus genome was identified in one of the samples.ConclusionsOverall this study demonstrated the application of whole genome sequencing as an important tool in molecular characterization of noroviruses.