Identification of spring viraemia of carp virus (SVCV) by combined RT-PCR and nested PCR

Diseases of Aquatic Organisms

I. Psikal¹  M. Koutná¹  T. Vesely¹  J. Hulová¹ 

关键词: RT-PCR;    SVCV;    Rhabdovirus;    Carp;    Sequence analysis;   

DOI  :  10.3354/dao055229

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

ABSTRACT: A combination of single-tube reverse transcription (RT)-PCR and nested PCR was used to identify spring viraemia of carp rhabdovirus (SVCV) in infected cell cultures and fish tissues. Two pairs of specific primers (external and internal)were selected from the glycoprotein gene sequence. A specific product of 470 bp was amplified from RNA derived from 34 SVCV isolates including the reference strain (Fijan), using RT-PCR with the external primers. The subsequent PCR using the internalprimers yielded a specific product of 141 bp in all cases. No PCR product was obtained following attempts to amplify RNA derived from other fish viruses including pike fry rhabdovirus (PFRV), or from non-infected cells. The identity of the cDNA wasconfirmed by direct sequencing. PCR sensitivity in the cell-culture system was assessed as about 10^-1 TCID₅₀ ml^-1. PCR was further used for the detection of SVCV in 14 clinical samples. Nested PCR allowed us to diagnosethe infection in all clinical samples in which SVCV infection was demonstrated by electron microscopy and ELISA. PCR amplification of the SVCV glycoprotein (G) gene is a potential method for rapid diagnosis of spring viraemia of carp; however, it isnecessary to verify the method in a higher number of clinical tissue samples. PCR can now be added to current confirmatory diagnostic methods, for determination of SVCV in cell culture. Sequencing of RT-PCR products performed for 7 SVCV isolates (4 Czech,2 Hungarian, and 1 isolate of unknown origin) revealed a high degree of homogeneity of the G gene region with that of the previously sequenced Fijan strain. The highest nucleotide variability (97.4 to 98.1% nucleotide similarity) was found between theHungarian and the other isolates. Knowledge of genetic differences among SVCV isolates will be useful in the development of diagnostic methods and elaboration of vaccination programmes.

【 授权许可】

Unknown