UDC 636.4:619:578.835.2:636.085:577.2:57.083
doi: 10.15389/agrobiology.2014.4.75eng
ТHE USE OF MEMBRANE FILTERS IN DETECTION OF THE ASF VIRUS DNA IN THE FEED COMPOUND BY RT-PCR
D.A. Kudryashov, S.A. Katorkin, E.V. Aronova, O.N. Burdinskaya, Yu.P. Morgunov, I.Kh. Gazaev, S.Zh. Tsybanov, D.V. Kolbasov
All-Russian Institute of Veterinary Virology and Microbiology, Russian Academy of Agricultural Sciences,
Pokrov, Petushinskii Region, Vladimir Province, 601120 Russia,
e-mail kolbasovdenis@gmail.com, is.gazaev@mail.ru
Received March 31, 2014
For African swine fever (ASF) virus an alimentary transmission way is known to be characteristic. In particular, the infection occurs because of use of the contaminated domestic and slaughtering wastes or the feed compounds originated from epizootic region which have not been duly controlled, or in case the package used for feed transportation were not treated against African swine virus. Under feed compound analysis by real-time PCR (RT-PCR), the samples must be free from large particles and chemical substances which can inhibit the polymerase reaction and influence the sorbent and membrane properties during nucleic acid isolation. We aimed to improve the feed compound processing technique by using membrane filters to prepare the samples for further RT-PCR indication of ASF virus. In the experiments, the feed was artificially contaminated by blood of a swine previously infected by the ASF virus Stavropol 2009 strain, the titer of 5.0 lg HAD50/cm3. Also the dilutions of 2.0, 3.0 and 4.0 HAD50/cm3 were tested. During processing, the crud filtration, freezing-defrostation procedure, centrifugation and separation on membrane filters (450 mm) were applied. Then the ASF virus DNA isolation and PCR-RT were conducted using the developed test-system and procedure. It was found out that the ASF virus DNA can be detected by the proposed method in the feed compound when the titer of virus used for artificially contaminated is at least 3.0 lg HAD50/cm3. So, the modifying procedure of fodder sample preparation described hereinabove is effective to obtain purified and concentrated material for further ASF virus DNA analysis.
Keywords: African swine fever, ASF, African swine fever virus, feed compound, sample preparation, membrane filters, RT-PCR.
REFERENCES
1. Sánchez-Vizcaíno J.M., Mur L. African swine fever diagnosis update. Dev. Biol. (Basel), 2013, 135: 159-165. CrossRef
2. Nigsch A., Costard S., Jones B.A., Pfeiffer D.U., Wieland B. Stochastic spatio-temporal modelling of African swine fever spread in the European Union during the high risk period. Prev. Vet .Med., 2013, 108(4): 262-275. CrossRef
3. Atuhaire D.K., Afayoa M., Ochwo S., Mwesigwa S., Mwiine F.N., Okuni J.B., Olaho-Mukani W., Ojok L. Prevalence of African swine fever virus in apparently healthy domestic pigs in Uganda. BMC Vet. Res., 2013, 9: 263. CrossRef
4. De Carvalho Ferreira H.C., Backer J.A., Weesendorp E., Klinkenberg D., Stegeman J.A., Loeffen W.L. Transmission rate of African swine fever virus under experimental conditions. Vet. Microbiol., 2013, 165(3-4): 296-304. CrossRef
5. Gómez-Villamandos J.C., Bautista M.J., Sánchez-Cordón P.J., Carras-
co L. Pathology of African swine fever: the role of monocyte-macrophage. Virus Res., 2013, 173(1): 140-149. CrossRef
6. Bakulov I.A. Afrikanskaya chuma svinei [African swine fever]. Moscow, 1969.
7. Kozlova D.I., Beskhlebnov V.A. Sovremennye problemy afrikanskoi chumy svinei [Current problems of African swine fever]. Moscow, 1980.
8. Atrazheva T.A. V knige: Puti povysheniya kachestva produktov zhivotnovodstva i ikh veterinarno-sanitarnaya otsenka [In: The ways to improve quality of livestock products and their veterinary and sanitary control]. Kiev, 1981.
9. GOST 13496.0-80 «Kombikorma, syr'e, metody otbora prob» [State Standard of RF 13496.0-80 «Combined fodders, raw materials, sampling procedure»]. Moscow, 1980.
10. Rukovodstvo po indikatsii osobo opasnykh boleznei sel'skokhozyaistvennykh zhivotnykh v ob"ektakh veterinarnogo nadzora i okruzhayushchei sredy /Pod redaktsiei N.A. Lagutkina, V.N. Smirnova, V.V. Kurinnova, S.Zh. Tsybanova [Indication of particularly dangerous infections under veterinary inspection in livestock and environment: guidance. N.A. Lagutkin, V.N. Smirnov, V.V. Kurinnov, S.Zh. Tsybanov (eds.)]. Moscow, 2000.
11. Gazaev I.Kh., Elsukova A.A., Sindryakova I.P., Tsybanov S.Zh., Kolbasov D.V. V sbornike: Molekulyarnaya diagnostika-2010 [In: Molecular diagnostics-2010]. Moscow, 2010: 83-85.
12. Boom R., Sol C.J., Salimans M.M. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol., 1990, 28(3): 495-503.
13. Lucas A., Haag J., Larenaudie B. La peste porcine africaine. Collection de monographies. Paris, 1967.
