Medycyna Wet. 67 (3), 168-171, 2011
Niedbalski W., Kęsy A.
Methods of DNA polymerization in vitro for the diagnosis of bluetongue
DNA polymerization techniques in vitro have become some of the most widely used methods of diagnosing epizootic diseases of livestock, including bluetongue (BT). The article reviews the RT-PCR assays (conventional and quantitative) recently developed and used for the detection of viral RNA in biological samples. Several of the most conserved BTV genome segments have been used as targets for RT-PCRs: genome segment 5, encoding the highly conserved non-structural protein NS1; segment 7, encoding the BTV surface structural protein VP7; segment 10, encoding NS3/3a; and segment 1, encoding VP1, the viral RNA-dependent RNA polymerase. Detection of BTV RNA in different tissues, such as blood, lymph nodes and spleen is also possible by many conventional RT-PCR assays. These methods, however, require agarose gel electrophoresis, which is rather laborious and time-consuming. The real-time RT-PCR (rRT-PCR) is faster, easier and more sensitive than the traditional gel-based RT-PCR methods. This format eliminates the need to open the reaction tube during and after amplification, greatly reducing the risk of contamination. Since August 2006, when the first BT outbreaks caused by serotype 8 occurred in North-Western Europe, many rRT-PCR assays have been developed and are currently routinely used in countries across Europe.
Keywords: bluetongue, diagnostic methods, RT-PCR and real-time RT-PCR