1. Role of the sample weight in norovirus detection in oysters by using molecular techniques Student: Noor Meersseman Work placement employer: Leena Maunula Work placement mentor: Kirsi Söderberg Work placement supervisor: Griet Vanbillemont

2. Overview presentation 1.Introduction: background and aims 2.Methods 2.1 Sample collection and processing of the oysters 2.2 RNA extraction 2.3 RT-qPCR 3.Results 3.1 RT-PCR inhibition 3.2 Extraction efficiency 3.3 Sample quantification 4.Conclusion 5.Future 17/06/2015 2

3. 1.Background and aims Risks of raw oysters: norovirus Cause of non-bacterial gastroenteritis Several outbreaks each year Analyze raw oysters for the presence of noroviruses Calculate amount of genome copies in positive samples Determine importance of weight/ sample size 17/06/2015 3

4. 2.Methods 17/06/2015 4 10 batches, each 6 oysters 5 oysters1 oyster Virus extraction from digestive gland RNA extraction using NucliSENS miniMAG RT-qPCR (NoV GI + NoV GII)

5. 2.Methods 2.1 Sample collection and processing of the oysters Crassostrea gigas or Pacific oyster 10 weeks  10 batches  60 oysters in total Virus extraction: 17/06/2015 5 Digestive gland Mengo virus control Proteinase K solution Incubation Centrifugation Process control

6. 2.Methods 2.2 RNA extraction Supernatant + lysisbuffer NucliSENS miniMAG extraction machine + reagents Silica-containing magnetic beads 17/06/2015 6

7. 2.Methods 17/06/2015 7

8. 2.Methods 2.3 RT-qPCR Principle: One-step PCR  RT step + PCR reaction in one single tube RNA  cDNA (Reverse transcription enzyme) cDNA  exponential amplification Real-time PCR  amplification is followed during run (probes) 17/06/2015 8

9. 2.Methods 2.3 RT-qPCR Mengo virus, NoV GI and NoV GII Specific primers and probes Rotor Gene - ILS15  Initial activation: 55°C – 60 min  Second activation: 95°C – 15 min  45 cycles: 95°C – 15 sec, 60°C – 60 sec, 65°C – 60 sec Also included in run:  EC RNA (RT-PCR inhibition)  dsDNA (quantification) 17/06/2015 9

10. 3.Results 3.1 RT-PCR inhibition Sample + EC RNA C t values Above 75% = Fail 17/06/2015 10

11. 3.Results 17/06/2015 11 GIGII

12. 3.Results 3.2 Extraction efficiency Sample C t values from Mengo virus PCR runs Below 1% = Fail 2/ 19 failed Extraction repeated with batch 3, 7 and 9 17/06/2015 12

13. 3.Results 3.3 Sample quantification: all results GI and GII 14/19 positive for GI (74%) 3 also positive for GII 17/06/2015 13 GI GII

14. 3.Results 3.3 Sample quantification: results according to batch number 17/06/2015 14

15. 4.Conclusion Detection of noroviruses successful in both sample types Quantification: amount of detected genome copies/g slightly higher in 1-oyster samples Inhibition: better results using 5-oyster method Extration efficiency: better mean result for 5-oyster samples 17/06/2015 15

16. 5.Future Further research is needed Bigger amount of samples Winter  summer 17/06/2015 16

17. Role of the sample weight in norovirus detection in oysters by using molecular techniques 17/06/2015 17 Thank you for your attention! Any questions?