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Primers used for the detection of the carp oedema virus P4a protein in a real-time PCR
| Primer name | Primer sequence | Reference |
|---|---|---|
| CEV qFor1 | 5′-AGTTTTGTAKATTGTAGCATTTCC-3′ | |
| CEV qRev1 | 5′-GATTCCTCAAGGAGTTDCAGTAAA-3′ | 12 |
| CEV qProbe1 | 5′-AGAGT TTGTTTCTTGCC ATACAAACT-3′ |
Summary of carp tissue samples (kidney, spleen, gills and skin) subjected to in situ hybridisation
| Sample collection site | Study material | Species | Symbol |
|---|---|---|---|
| Farm 2 | kidney, spleen | Carp (Cyprinus carpio) | DC1 |
| Farm 2 | gills, skin | Carp (Cyprinus carpio) | DC1 |
| Farm 2 | kidney, spleen | Carp (Cyprinus carpio) | DC2 |
| Farm 2 | gills, skin | Carp (Cyprinus carpio) | DC2 |
| Farm 2 | kidney, spleen | Carp (Cyprinus carpio) | DC4 |
| Farm 2 | gills, skin | Carp (Cyprinus carpio) | DC4 |
| Farm 2 | kidney, spleen | Carp (Cyprinus carpio) | DC7 |
| Farm 2 | gills, skin | Carp (Cyprinus carpio) | DC7 |
| Farm 2 | kidney, spleen | Carp (Cyprinus carpio) | DC8 |
| Farm 2 | gills, skin | Carp (Cyprinus carpio) | DC8 |
| Farm 2 | kidney, spleen | Carp (Cyprinus carpio) | DC9 |
| Farm 2 | gills, skin | Carp (Cyprinus carpio) | DC9 |
| Farm 9 | kidney, spleen | Carp (Cyprinus carpio) | 9SK |
| Farm 9 | gills, skin | Carp (Cyprinus carpio) | 9SK |
List of positive samples in carp obtained from kidney, spleen, gills and skin using in situ hybridisation
| Sample collection site | Code | Kidney | Spleen | Gills | Skin |
|---|---|---|---|---|---|
| GR 2 | DC1 | − | − | + | + |
| GR 2 | DC2 | + | − | + | + |
| GR 2 | DC4 | − | − | + | − |
| GR 2 | DC7 | + | − | + | − |
| GR 2 | DC8 | + | − | + | − |
| GR 2 | DC9 | + | − | + | − |
| GR 9 | 9SK | + | − | + | + |
Carp oedema virus–positive samples in common carp (Cyprinus carpio) detected using real-time PCR
| No. | Sample code | Sample collection site | Threshold cycle |
|---|---|---|---|
| 1 | DC 4 | Farm 2 | 36.67 |
| 2 | DC 7 | Farm 2 | 28.26 |
| 3 | DC 8 | Farm 2 | 24.44 |
| 4 | DC 9 | Farm 2 | 23.47 |
| 5 | DC 10 | Farm 2 | 25.08 |
| 6 | DC 11 | Farm 2 | 24.55 |
| 7 | DC12 | Farm 2 | 24.82 |
| 8 | DC 5 | Farm 5 | 28.2 |
Primers used for the detection of the carp oedema virus P4a protein in conventional and nested PCRs
| Primer name | Primer sequence | Product size (base pairs) | Reference |
|---|---|---|---|
| CEV qFor1 | 5′-ATGGAGTATCCAAAGTACTTAG-3′ | 528 | |
| CEV for B | |||
| CEV rev J | 5′-CTCTTCACTATTGTGACTTTG-3′ | 528 | 12 |
| CEV for B - int | 5′-GTTATCAATGAAATTTGTGTATTG-3′ | 478 | |
| CEV rev J - int | 5′-TAGCAAAGTACTACCTCATCC-3′ | 478 |
Composition of the PCR mixture used for detection of the carp oedema virus P4a protein in a second nested PCR
| Deionised water (PCR grade) | GoTaq G2 Green Master Mix | Forward primer | Reverse primer | Template DNA |
|---|---|---|---|---|
| 6.5 mL | 12.5 mL | 0.5 mL | 0.5 mL | 5 mL |
Composition of the real-time PCR mixture for detection of carp oedema virus P4a protein
| Distilled water (PCR grade) | GoTaq G2 Green Master Mix | Forward primer | Reverse primer | TaqMan probe | Template DNA |
|---|---|---|---|---|---|
| 6.25 mL | 12.5 mL | 0.5 mL | 0.5 mL | 0.25 mL | 5 mL |