Wpływ utrwalaczy organicznych na stabilność materiału genetycznego DNA herpeswirusa koi (KHV)

• Autorzy: Borzym E. , Maj J. , Matras M.

Warianty tytułu

EN

Impact of organic fixatives on the stability of the DNA genetic material of the koi herpes virus (KHV)

• Języki publikacji: PL

Abstrakty

EN

Studies to identify the koi herpes virus using the PCR are continuously developed and introduced into routine diagnostics. Well-selected ways of storing tissues that are later used for DNA extraction and for the amplification reaction are critical points in methods using molecular biology techniques. The objective of the studies was to determine the impact of the fixating properties of the two most frequently used organic compounds, ethanol 96% and 80%, and isopropanol, on the stability of the nucleic acid isolated and on the preservation of the koi herpes virus in selected tissues and organs of carps. The experimental fish were infected via a bath in a small 20 l aquarium, where 5 ml of a KHV reference strain suspension had been added to the water. The presence and stability of the total DNA isolated from tissues and organs (skin, brain, kidney, gills) was checked using PCR with TK primers designed by Bercovier, and a product of 409 bp was obtained. Primers amplifying a gene fragment of beta-actin, which is found in smooth muscles and non-muscle cells of fish, were used as isolation control in multiplex PCR. On the basis of the studies it was observed that just on the day the samples were taken from the internal organs of fish and a month later, when they were collected from the archived organs, a product of 409 bp and 259 bp was obtained in all the samples analysed. The best organic fixative of organs and tissues sampled for molecular tests to identify KHV is ethanol 96%, even after half a year of archiving. Ethanol 80% and isopropanol also ensure sufficient fixation for up to a month. The most stable virus DNA is isolated from fish skin and gills.

Słowa kluczowe

PL

ryby; choroby zwierzat; choroby wirusowe; diagnostyka; izolacja wirusow; wirus KHV; tkanki zwierzece; przechowywanie; utrwalacze organiczne; etanol; izopropanol; wlasciwosci utrwalajace; DNA wirusowe; stabilnosc

• Wydawca: -
• Czasopismo: Medycyna Weterynaryjna
• Rocznik: 2012
• Tom: 68
• Numer: 06
• Opis fizyczny: s.359-361,fot.,tab.,bibliogr.

Twórcy

autor

Borzym E.

• Zakład Chorób Ryb, Państwowy Instytut Weterynaryjny - Państwowy Instytut Badawczy, Al.Partyzantów 57, 24-100 Puławy

autor

Maj J.

autor

Matras M.

Bibliografia

• 1.Anon.: OIE Manual of diagnostic tests for aquatic animals. World Organization for Animal Health, Paris 2009.
• 2.Bercovier H., Fishman Y., Nahary R., Sinai S., Zlotkin A., Eyngor M., Gilad O., Eldar A., Hedrick R. P.: Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis. BMC Microbiol. 2005, 5, 13.
• 3.Gilad O., Yun S., Zagmutt-Vergara F. J., Leutenegger C. M., Bercovier H., Hedrick R. P.: Concentrations of a Koi herpesvirus (KHV) in tissues of experimentally infected Cyprinus carpio koi as assessed by real-time TaqMan PCR. Dis. Aquat. Org. 2004, 60, 179-187.
• 4.Gray W. L., Mullis L., LaPatra S. E., Groff J. M., Goodwin A.: Detection of koi herpesvirus DNA in tissues of infected fish. J. Fish. Dis. 2002, 25, 171-178.
• 5.Kempter J., Kiełpiński M., Panicz R., Sadowski J.: Określenie nosicielstwa i podatności na infekcje koi-herpes-virusem wybranych gatunków ryb karpiowatych i ich krzyżówek pochodzących z wód otwartych i obiektów hodowlanych położonych w zlewni Odry. Wyd. AR, Szczecin 2008.
• 6.Kilpatrick C. W.: Noncryogenic reservation of mammalian tissues for DNA extraction: an assessment of storage methods. Bioch. Gen. 2002, 40, 53-62.
• 7.Smith L. J., Braylon R. C., Nutkis J. E., Edmundson K. B., Downing J. R., Wakeland E. K.: Extraction of cellular DNA from human cells and tissues fixed in ethanol. A. Bioch. 1987, 160, 135-138.