Abstract

A conventional PCR based on targeting the thymidine kinase (tk) gene has been shown to be specific to CyHV3¹, but the PCR method can also lead to false positive results in some samples. The authors developed a real-time PCR to detect CyHV3 specifically and to overcome some false positive reactions of the conventional tk gene PCR in a survey of CyHV3 infection in Korea. The real-time PCR, based on targeting the CyHV3 ribonucleotide reductase gene, could detect the targeting gene to the 1 fg level and was not detected in several kinds of normal fish tissue, including koi tissue and non-template controls. We surveyed for CyHV3 infection using both the tk gene PCR and the real-time PCR on the imported ornamental fish, farm-cultured koi carp and wild common carp in Korea. We screened 463 samples composed of 400 samples of 32 species of ornamental fish imported from 4 countries into Korea, 42 samples of koi carp from 5 koi farms and 21 samples of common carp from 10 different environmental habitats. There were some positive reactions on the ornamental fish and one farm cultured koi in the tk gene PCR, but the real-time PCR showed no reaction. Although real-time PCR did not amplify the CyHV3 in all the samples, we did expand the investigation of CyHV3 with more experiments on the koi farm samples. The koi farm samples were inoculated into CCB cell line and then checked for CyHV3 amplification.⁵ The same samples were also inoculated into healthy koi through the abdominal cavity.^3,4 From the results we reconfirmed the koi farm samples were negative. In the survey of CyHV3 infection in natural habitat common carp, both of the two detection methods showed negative results. The combined tk gene PCR with real-time PCR were useful tools to differentiate the CyHV3² from CyHV1 and 2 and overcome some false positive reactions for surveying of CyHV3 infection. We report here that in Korea, the results of the survey of CyHV3 infection were negative.

Acknowledgements

This project was supported by a grant from the National Veterinary Research and Quarantine Service; Ministry of Food, Agriculture, Forestry and Fisheries; Republic of Korea.

References

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