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
Overview presentation
2.1 Sample collection and processing of the oysters
2.2 RNA extraction
2.3 RT-qPCR
17/06/2015
1. Introduction: background and aims
2. Methods
3. Results
3.1 RT-PCR inhibition
3.2 Extraction efficiency
3.3 Sample quantification
4. Conclusion
5. Future
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• Risks of raw oysters: norovirus
• Cause of non-bacterial gastroenteritis
• Several outbreaks each year
17/06/2015
1. Background and aims
• Analyze raw oysters for the presence of noroviruses
• Calculate amount of genome copies in positive samples
• Determine importance of weight/ sample size
3
2. Methods
5 oysters
1 oyster
17/06/2015
10 batches, each 6 oysters
Virus extraction from digestive gland
RNA extraction using NucliSENS miniMAG
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RT-qPCR (NoV GI + NoV GII)
2.1 Sample collection and processing of the
oysters
• Crassostrea gigas or Pacific oyster
• 10 weeks  10 batches  60 oysters in total
• Virus extraction:
Digestive gland
17/06/2015
2. Methods
Process control
Mengo virus control
Proteinase K solution
Incubation
Centrifugation
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2. Methods
• Supernatant + lysisbuffer
• NucliSENS miniMAG extraction machine + reagents
• Silica-containing magnetic beads
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2.2 RNA extraction
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2. Methods
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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)
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2. Methods
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2. Methods
• Mengo virus, NoV GI and NoV GII
• Specific primers and probes
• Rotor Gene - ILS15
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2.3 RT-qPCR
 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)
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3. Results
• Sample + EC RNA Ct values
• Above 75% = Fail
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3.1 RT-PCR inhibition
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3. Results
Sample (Un-)diluted % inhibition Fail
1
1:1
56,48
1
1:1
30,84
2
1:1
42,16
2
1:1
38,84
3
1:1
33,79
3
1:1
12,20
4
1:1
89,12 X
4
1:1
45,13
0,00
5
1:1
1:10
5
1:1
0,00
6
1:1
0,00
6
1:1
66,46
7
1:1
40,82
7
1:1
100,00 X
8
1:1
73,35
1:10
33,06
9
1:1
33,13
8
1:1
67,93
10
1:1
58,69
9
1:1
67,69
11
1:1
46,10
10
1:1
50,64
1:10
52,64
11
1:1
44,00
1:1
22,80
12
1:1
28,96
1:10
21,17
13
1:1
18,23
1:1
51,37
14
1:1
37,88
0,00
15
1:1
18,70
13
1:10
12
1:10
0,00
14
1:1
28,55
16
1:1
63,74
15
1:1
3,31
17
1:1
47,72
16
1:1
41,20
18
1:1
43,21
17
1:1
44,66
19
1:1
95,61 X
1:10
0,00
1:10
24,12
18
1:1
9,59
20
1:1
58,26
19
1:1
29,03
21
1:1
4,56
20
1:1
53,90
22
1:1
57,70
21
1:1
52,53
23
1:1
39,36
22
1:1
42,60
24
1:1
51,98
23
1:1
47,42
24
1:1
17,30
1 No RT-PC R inhibtion value
0,00
was obtained for this sample
1:10
GII
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GI
Sample (Un-)diluted % inhibition Fail
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3. Results
•
•
•
•
Sample Ct values from Mengo virus PCR runs
Below 1% = Fail
2/ 19 failed
Extraction repeated with batch 3, 7 and 9
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3.2 Extraction efficiency
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3. Results
• 14/19 positive for GI (74%)
• 3 also positive for GII
GI
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3.3 Sample quantification: all results GI and GII
GII
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3.3 Sample quantification: results according to
batch number
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3. Results
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• 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
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4. Conclusion
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• Further research is needed
• Bigger amount of samples
• Winter  summer
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5. Future
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17/06/2015
Role of the sample weight in
norovirus detection in
oysters by using molecular
techniques
Thank you for your attention!
Any questions?
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