qpcr detection method for foodborn pathogens Vibrio parahaemolytiucus and Norovirus Dr. Ahmed Siah Research Scientist

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1 qpcr detection method for foodborn pathogens Vibrio parahaemolytiucus and Norovirus Dr. Ahmed Siah Research Scientist

2 Outline BC CAHS services Development of a QPCR for V. parahaemolyticus Spiked experiment Test comparison Testing norovirus in oysters Genogroups Testing method Summary

3 Who are BC CAHS? Non-profit society Stakeholders, not shareholders Fee for services model Research and diagnostics laboratory Fisheries and aquaculture In operation for 12+ years Employs local HQP 13 FTE + two seasonal PT 4 PhD, 2 MSc, 6 BSc + Tech Dipl Co-op students

4 Timeline of Growth

5 Development of a qpcr detection tool for Vibrio parahaemolyticus

6 Objectives 1. Develop a qpcr assay to detect Vibrio parahaemolyticus 2. Screening field samples 3. Relationship between qpcr and MPN

7 qpcr -> thermolabile hemolysin (tlh) target (Jones et al., 2016)

8 qpcr -> ABC transporter target (Liu et al., 2012)

9 qpcr -> phosphatidyl serine synthase (pss) target (CAHS)

10 Sample 10 ml -> Filter 0.22um -> Place filter in 30 ml APW -> Incubation 30C for hours (Turner et al., 2014) Plankton testing pss ABC tlh

11 Experimental 500g (~10 oysters) spiked with 1e+6, 1e+8, 1e+10, 1e+12 design 50g + 50 ml PBS 20g + 80 ml PBS 100ul + 10 ml APW 10ul + 10 ml APW 1ul + 10 ml APW Incubate 35C for hours plating qpcr plating plating qpcr

12 Growth curve experiment

13 Spike experiment Log CFU/mL = -4.1e+08tlh+7.4e+09 R square = 0.81 p< Log CFU/mL = -4.2e+08tlh+7.6e+09 R square = 0.83 p< Log CFU/mL = -3.8e+08tlh+6.9e+09 R square = 0.53 p<0.01 Log CFU/mL = -3.7e+08tlh+6.8e+09 R square = 0.57 p<0.005

14 Field samples

15 Next steps Spiked experiment with lower concentrations of Vpara Perform simultaneously qpcr and classic bacteriology screening Validate the qpcr test on field samples Work with regulatory agencies for implementation the assay

16 Funding Partner

17 Testing Norovirus in oysters

18 NoroVirus (NoV) Family Calciviridae -> Latin Calyx -> cup ~40 nm virus like particles Small round-structured viruses Non enveloped icosahedral No cell culture available Single stranded positive-sense RNA genome ~7.6kb Robilotti et al., 2015

19 Genogroups Zheng et al.,2006

20 High Variability Donaldson et al., 2010

21

22 qpcr Assays Genogroup GI Genogroup GII

23 Challenges Low levels of viruses Variability in virus or nucleic acid extraction Presence of inhibitors ie polysaccharides, fatty acid and protein should be removed

24 NoV RNA Extraction 2 g of digestive gland (~6 oysters) that has receptor. Houde et al., OPFLP

25 European Standard Methods (Lees 2010) Baylor Method (Atmar et al., 1995) Proteinase K digestion Method (Jothikumar et al., 2005) -> Simpler method Purification and concentration -> Boom method (Boom et al., 1990) utilize Guanidine isothiocyanate (GITC) to denature viral coat proteins in combination with silica particles (Nuclisens) to bind released nucleic acid which is then purified through successive washing stages before elution.

26 Fluorescence Real Time PCR based techniques Plateau Phase CAHS 2012 QuantStudio 72k Flex 72,000 reactions CAHS 2007 ABI 7500 Fast 384 reactions Ct value PCR Cycles

27 Conclusions NoV (family Calciviridae)-> Agent for human gastroenteritis Non-envelopped, icosahedral, single stranded positive-sense RNA genome Genetically and antigenically diverse Genogroups based on complete capsid gene 5 genogroups: I, II, IV human strains III, V infect only animals

28 Next Phases Phase 1: To implement an abbreviated version of the ISO protocol Phase 2: Epidemiological studies

29 Take away Early detection will allow a better mitigation of pathogen outbreak Development of a rapid and sensitive testing tool -> Epidemiological studies qpcr based methods meet these requirement mainly when viral cell cultures are not available BC CAHS has the capacity and the experience in implementing qpcr screening tools for both Vibrio parahaemolyticus and Norovirus

30 The Team Dr. Jim Powell, CEO Ms. Tina Podlasly, Corporate Manager Dr. Ahmed Siah, Research Scientist Dr. Wyth Marshall, Research Scientist Dr. Shannon Balfry Ms. Elan Downey, Research Assistant Ms. Debbie Collins, Laboratory Technician Ms. Heather McDonald, Research Assistant Ms. Zina Richmond, Senior Laboratory Technician Mr. Robert Johns, Microbiology Technician Ms. Kim Bull, Laboratory Technician