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Real time PCR allows simultaneous amplification and quantification of PCR products.
ДThe amplified fragment of CSF DNA can be detected using a molecular beacon (a short chemically synthesised single-stranded oligonucleotide (25 b.p.)) complementary to the inner part of the amplified fragment of CSF virus that carries a fluorescent label at the 5'-end and a fluorescence quencher at the 3'-end. The beacon undergoes a conformational reorganization in the course of PCR as the specific fragment of the viral nucleic acid is amplified and forms a hybrid complex with the beacon, spatially separating the fluorescent label and quencher, thus exposing the fluorescent label. By measuring the fluorescence intensity, the results of PCR amplification can be visualized without gel electrophoresis.
By measuring the fluorescence intensity, the results of PCR amplification can be visualized without gel electrophoresis.
A major advantage is that reaction tubes stay closed during fluorescence measurements, which prevents contamination of the PCR products.
The test system allows differentiation between CSF virus and other pestiviruses such as bovine viral diarrhoea virus. Among the advantages of the test system is the capacity to detect the virus after general vaccination. The test system, however, should only be used two weeks after vaccination, i.e. after the elimination of the vaccine virus in the blood.
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