Real time PCR allows simultaneous amplification and quantification of PCR products.
Products of amplification can be differentiated using molecular beacons (short chemically synthesised single-stranded oligonucleotides 22 and 25 b.p. long) complementary to the inner part of the amplified fragment of either M. bovis or M.tuberculosis DNA fragments. Both beacons are 5'-end labelled with fluorescent dyes and 3'-labelled with quenchers. The beacons undergo a conformational reorganization in the course of PCR, as the PCR products are amplified and form a hybrid with the beacons, 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.
A major advantage is that reaction tubes stay closed during fluorescence measurements, which prevents contamination of the PCR products.
Practically, the test system consists of two kits, one of which allows detection of both M. bovis and н. tuberculosis, whereas the other one is designed for detection of M. tuberculosis only.
The test system also allows differentiation between the two pathogenic mycobacteria and the so called non-typical mycobacteria that cause no pathology.
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