Assessing tropism and genetic traits of carp oedema virus isolates to enhance detection strategies

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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