Nukleinsäure-basierte diagnostische Untersuchungen zum Nachweis von Pestiviren

For the confirmation and eradication of epizootic diseases, fast, sensitive, specific and reliable assays play a crucial role. For the direct detection of the different pathogens, novel molecular methods based on amplification of parts of the genome are more and more used. The most common method since several years is polymerase chain reaction (PCR). PCR technique allows the fast and highly sensitive detection of specific DNAs or RNAs. However, conventional PCRs are problematic concerning the risk of contamination, and control of specificity as well as quantification of genome copies in the samples are difficult. Therefore, an enhanced PCR technology, the so-called real-time PCR becomes widely accepted for the detection of infectious diseases. In real-time PCR, the amplicons are detected using specific probes labeled with fluorescence colors. Therefore, PCR tubes need not to be opened, effectively reducing the risk of cross-contamination. In addition, real-time PCR allows exact quantification of genome copy numbers, since the change in fluorescence signaling is linked to the number of genome copies in the tested sample. The binding of the specific probe also ensures exclusion of false positive signals due to unspecific amplifications. With the combination of different fluorescence channels, several amplicons can be detected simultaneously and multiplex protocols, inlcuding internal controls, can be used. Here we present data about real-time RT-PCRs for the detection of pestiviruses, especially bovine viral diarrhea virus. Newly developed protocols, experiences with different sample materials as well as the investigation of pooled samples and samples within the "diagnostic gap" will be demonstrated and discussed.