Разработка методики количественной real-time ПЦР для идентификации вируса инфекционного ринотрахеита крупного рогатого скота
2009
Maksimovich, V.V. | Krasochko, P.P. | Kvach, S.V., Vitebsk State Academy of Veterinary Medicine (Belarus)
Data on working out a quantitative polymerase chain reaction (PCR) for revealing of virus of infectious bovine rhinotracheitis realized in the conditions of the Republic of Belarus was presented. The development of the qualitative PCR was realized in the following stages: analysis of viral genome and selection of primers; synthesis of primers and control of their specificity; optimization of conditions for PCR; obtaining of positive control and determination of its concentration; obtaining and testing of probe and optimization of its concentration for PCR; testing of the developed method and determination of sensitivity. The obtained method made it possible to determine not only presence of infectious bovine rhinotracheitis, but also its initial amount in sample. Due to application of probe constructed in accordance with molecular beacon technology the presented method proved to be highly specific. That was connected with the fact that fluorescence was registered only when the probe connected to complementary part of DNA, in other case the result was negative. Research results showed that the sensitivity of the given procedure made it possible to define in a sample presence of a virus with concentration 2 lg that corresponded to 2 DNA copies
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