Detection of spring viremia of carp virus isolates by hybridization with non-radioactive probes and amplification by polymerase chain reaction.
For detection of spring viremia of carp virus (SVCV) DNA probes have been constructed using the reverse transcription-polymerase chain reaction (RT-PCR) amplification technique and cDNA cloning in plasmid and phage vectors. The specific primers for amplification of SVCV M and G genes were chosen and synthesized. Studies were carried out to establish the sensitivity and specificity of viral RNA detection in infected cell culture and pathogenic material from fish by the use of non-radioactive probes and RT-PCR. The efficiency of amplification with primers, complementary to the genome of the reference Fijan strain, was estimated in RT-PCR experiments with two SVCV strains. Under the same conditions, the quantity of PCR products amplified from the M2 strain was less than that from the ZL4 strain, which implies that the latter is more similar to the reference European SVCV Fijan isolate. Using DNA probes and dot-blot hybridization, SVCV was tested in samples taken from different organs of artificially infected carp with clinical signs of acute disease. The virus could be detected most reliably in fish brain. In most cases the hybridization signal was positive with samples having a viral titer of not less than 10(5) TCID50/g.
Oreshkova
S F
SF
Research Institute of Bioengineering of State Research Centre of Virology and Biotechnology 'Vector', Koltsovo, Novosibirsk Region, Russia.
Shchelkunov
I S
IS
Tikunova
N V
NV
Shchelkunova
T I
TI
Puzyrev
A T
AT
Ilyichev
A A
AA
D000818:Animals; D019921:Biotinylation; D002347:Carps / Q000821:virology*; D015342:DNA Probes; D005393:Fish Diseases / Q000235:genetics / Q000821:virology*; D009693:Nucleic Acid Hybridization / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012209:Rhabdoviridae / Q000235:genetics* / Q000302:isolation & purification; D018353:Rhabdoviridae Infections / Q000235:genetics / Q000662:veterinary*; D012680:Sensitivity and Specificity
D015342:DNA Probes